Transgenic plants with enhanced agronomic traits

ABSTRACT

This invention provides recombinant DNA for expression of proteins that are useful for imparting enhanced agronomic trait(s) to transgenic crop plants. Also provided by this invention is transgenic seed for growing a transgenic plant having recombinant DNA in its genome and exhibiting an enhance agronomic trait, i.e. enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, enhanced tolerance to cold stress and/or improved seed compositions. Also disclosed are methods for identifying such transgenic plants by screening for nitrogen use efficiency, yield, water use efficiency, growth under cold stress, and seed composition changes. This invention also discloses a method of identifying the target genes of a transcription factor.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35USC § 119(e) of U.S. provisional application Ser. No. 60/713,150, filed Aug. 30, 2005, and incorporated herein by reference.

INCORPORATION OF SEQUENCE LISTING

Two copies of the sequence listing (Copy 1 and Copy 2) and a computer readable form (CRF) of the sequence listing, all on CD-Rs, each containing the text file named 38-21(53948)C_seqListing.txt, which is 33,136,640 bytes (measured in MS-WINDOWS) and was created on Aug. 30, 2006 are incorporated herein by reference.

INCORPORATION OF COMPUTER PROGRAM LISTING

One copy of the Computer Program Listing (Copy 1) and a computer readable form (CRF) containing folders hmmer-2.3.2 and 124pfamDir, all on CD-Rs are incorporated herein by reference in their entirety. Folder hmmer-2.3.2 contains the source code and other associated file for implementing the HMMer software for Pfam analysis. Folder 124pfamDir contains 124 Pfam Hidden Markov Models. Both folders were created on CD-R on Aug. 30, 2006, having a total size of 12,042,240 bytes (measured in MS-WINDOWS).

FIELD OF THE INVENTION

Disclosed herein are inventions in the field of plant genetics and developmental biology. More specifically, the present inventions provide transgenic seeds for crops, wherein the genome of said seed comprises recombinant DNA, the expression of which results in the production of transgenic plants with enhanced agronomic traits.

BACKGROUND OF THE INVENTION

Transgenic plants with enhanced agronomic traits such as increased yield, enhanced environmental stress tolerance, enhanced pest resistance, enhanced herbicide tolerance, improved seed compositions, and the like are desired by both farmers and consumers. Although considerable efforts in plant breeding have provided significant gains in desired traits, the ability to introduce specific DNA into plant genomes provides further opportunities for generation of plants with enhanced and/or unique traits. Merely introducing recombinant DNA into a plant genome doesn't always produce a transgenic plant with an enhanced agronomic trait. Thorough screening is required to identify those transgenic events that are characterized by the enhanced agronomic trait.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a map of plasmid pMON82060.

FIG. 2 is a map of plasmid pMON82053

FIG. 3 is a map of plasmid pMON99053

FIG. 4 is a map of plasmid pMON17730

SUMMARY OF THE INVENTION

This invention employs recombinant DNA for expression of proteins that are useful for imparting enhanced agronomic traits to the transgenic plants. Recombinant DNA in this invention is provided in a construct comprising a promoter that is functional in plant cells and that is operably linked to DNA that encodes a protein having at least one amino acid domain in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam domain names as identified in Table 11. In more specific embodiments of the invention the protein expressed in plant cells has an amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group of consensus amino acid sequences consisting of the consensus amino acid sequence constructed for SEQ ID NO: 194 and homologs thereof listed in Table 7 through the consensus amino acid sequence constructed for SEQ ID NO: 386 and homologs thereof listed in Table 7. In even more specific embodiments of the invention the protein expressed in plant cells is a protein selected from the group of proteins identified in Table 1.

Other aspects of the invention are specifically directed to transgenic plant cells comprising the recombinant DNA of the invention, transgenic plants comprising a plurality of such plant cells, progeny transgenic seed, embryo and transgenic pollen from such plants. Such plant cells are selected from a population of transgenic plants regenerated from plant cells transformed with recombinant DNA and that express the protein by screening transgenic plants in the population for an enhanced trait as compared to control plants that do not have said recombinant DNA, where the enhanced trait is selected from group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.

In yet another aspect of the invention the plant cells, plants, seeds, embryo and pollen further comprise DNA expressing a protein that provides tolerance from exposure to an herbicide applied at levels that are lethal to a wild type of said plant cell. Such tolerance is especially useful not only as an advantageous trait in such plants but is also useful in a selection step in the methods of the invention. In aspects of the invention the agent of such herbicide is a glyphosate, dicamba, or glufosinate compound.

Yet other aspects of the invention provide transgenic plants which are homozygous for the recombinant DNA and transgenic seed of the invention from corn, soybean, cotton, canola, alfalfa, wheat or rice plants.

In other important embodiments for practice of various aspects of the invention, the plants of this invention can be further enhanced with stacked traits, e.g., a crop having an enhanced agronomic trait resulting from expression of DNA disclosed herein, in combination with herbicide, disease, and/or pest resistance traits.

This invention also provides methods for manufacturing non-natural, transgenic seed that can be used to produce a crop of transgenic plants with an enhanced trait resulting from expression of stably-integrated, recombinant DNA for expressing a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam names identified in Table 11. More specifically the method comprises (a) screening a population of plants for an enhanced trait and a recombinant DNA, where individual plants in the population can exhibit the trait at a level less than, essentially the same as or greater than the level that the trait is exhibited in control plants which do not express the recombinant DNA, (b) selecting from the population one or more plants that exhibit the trait at a level greater than the level that said trait is exhibited in control plants, (c) verifying that the recombinant DNA is stably integrated in said selected plants, (d) analyzing tissue of a selected plant to determine the production of a protein having the function of a protein encoded by nucleotides in a sequence of one of SEQ ID NO:1-193; and (e) collecting seed from a selected plant. In one aspect of the invention the plants in the population further comprise DNA expressing a protein that provides tolerance to exposure to an herbicide applied at levels that are lethal to wild type plant cells and the selecting is effected by treating the population with the herbicide, e.g. a glyphosate, dicamba, or glufosinate compound. In another aspect of the invention the plants are selected by identifying plants with the enhanced trait. The methods are especially useful for manufacturing corn, soybean, cotton, alfalfa, wheat or rice seed.

Another aspect of the invention provides a method of producing hybrid corn seed comprising acquiring hybrid corn seed from a herbicide tolerant corn plant which also has stably-integrated, recombinant DNA comprising a promoter that is (a) functional in plant cells and (b) is operably linked to DNA that encodes a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam names identified in Table 11. The methods further comprise producing corn plants from said hybrid corn seed, wherein a fraction of the plants produced from said hybrid corn seed is homozygous for said recombinant DNA, a fraction of the plants produced from said hybrid corn seed is hemizygous for said recombinant DNA, and a fraction of the plants produced from said hybrid corn seed has none of said recombinant DNA; selecting corn plants which are homozygous and hemizygous for said recombinant DNA by treating with an herbicide; collecting seed from herbicide-treated-surviving corn plants and planting said seed to produce further progeny corn plants; repeating the selecting and collecting steps at least once to produce an inbred corn line; and crossing the inbred corn line with a second corn line to produce hybrid seed.

Another aspect of the invention provides a method of selecting a plant comprising plant cells of the invention by using an immunoreactive antibody to detect the presence of protein expressed by recombinant DNA in seed or plant tissue. Yet another aspect of the invention provides anti-counterfeit milled seed having, as an indication of origin, a plant cell of this invention.

Still other aspects of this invention relate to transgenic plants with enhanced water use efficiency or enhanced nitrogen use efficiency. For instance, this invention provides methods of growing a corn, cotton or soybean crop without irrigation water comprising planting seed having plant cells of the invention which are selected for enhanced water use efficiency. Alternatively methods comprise applying reduced irrigation water, e.g. providing up to 300 millimeters of ground water during the production of a corn crop. This invention also provides methods of growing a corn, cotton or soybean crop without added nitrogen fertilizer comprising planting seed having plant cells of the invention which are selected for enhanced nitrogen use efficiency.

DETAILED DESCRIPTION OF THE INVENTION

In the attached sequence listing:

SEQ ID NO:1-193 are nucleotide sequences of the coding strand of DNA for “genes” used in the recombinant DNA imparting an enhanced trait in plant cells, i.e. each represents a coding sequence for a protein;

SEQ ID NO:194-386 are amino acid sequences of the cognate protein of the “genes” with nucleotide coding sequence 1-193;

SEQ ID NO: 387-12580 are amino acid sequences of homologous proteins;

SEQ ID NO: 12581-12601 are nucleotide sequences of the elements in base plasmid vectors

SEQ ID NO: 12602 is a consensus amino acid sequence.

SEQ ID NO: 12603 is a nucleotide sequence of a base plasmid vector useful for corn transformation; and

SEQ ID NO: 12604 is a nucleotide sequence of a base plasmid vector useful for soybean transformation.

SEQ ID NO: 12605 is a nucleotide sequence of a base plasmid vector useful for cotton transformation.

SEQ ID NO: 12606 is the nucleotide sequence of plasmid PMON17730.

SEQ ID NO: 12607 is the nucleotide sequence of PHE0010424_PMON17730.

As used herein, a “transgenic plant” means a plant whose genome has been altered by the incorporation of exogenous DNA, e.g., by transformation as described herein. The term “transgenic plant” is used to refer to the plant produced from an original transformation event, or progeny from later generations or crosses of a plant so transformed, so long as the progeny contains the exogenous genetic material in its genome. “Exogenous DNA” means DNA, e.g., recombinant DNA, originating from or constructed outside of the plant including natural or artificial DNA derived from the host “transformed” organism of a different organism.

As used herein, “recombinant DNA” means DNA which has been a genetically engineered or constructed outside of a cell, including DNA containing naturally occurring DNA or cDNA, or synthetic DNA.

As used herein, a “functional portion” of DNA is that part which comprises an encoding region for a protein segment that is sufficient to provide the desired enhanced agronomic trait in plants transformed with the DNA activity. Where expression of protein is desired, a functional portion will generally comprise the entire coding region for the protein, although certain deletions, truncations, rearrangements and the like of the protein may also maintain, or in some cases improve, the desired activity. One skilled in the art is aware of methods to screen for such desired modifications and such functional portion of the protein is considered within the scope of the present invention.

As used herein, “consensus sequence” means an artificial, amino acid sequence of conserved parts of the proteins encoded by homologous genes, e.g., as determined by a CLUSTALW alignment of amino acid sequence of homolog proteins.

As used herein, “homolog” means a protein in a group of proteins that perform the same biological function, e.g., provide an enhanced agronomic trait in transgenic plants of this invention. Homologs are expressed by homologous genes which are genes that encode proteins with the same or similar biological function. Homologous genes may be generated by the event of speciation (see ortholog) or by the event of genetic duplication (see paralog). Orthologs refer to a set of homologous genes in different species that evolved from a common ancestral gene by specification. Normally, orthologs retain the same function in the course of evolution; and paralogs refer to a set of homologous genes in the same species that have diverged from each other as a consequence of genetic duplication. Thus, homologous genes can be from the same or a different organism. Homologous DNA includes naturally occurring and synthetic variants. For instance, degeneracy of the genetic code provides the possibility to substitute at least one base of the protein encoding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced from the gene to be changed. Hence, a polynucleotide useful in the present invention may have any base sequence that has been changed from SEQ ID NO:1 through SEQ ID NO: 193 by substitution in accordance with degeneracy of the genetic code. Homologs are proteins which, when optimally aligned, has at least 60% identity (say at least 70% or 80% or 90% identity) over the full length of a protein identified herein, or a higher percent identity especially over a shorter functional part of the protein, e.g., 70% to 80 or 90% amino acid identity over a window of comparison comprising a functional part of the protein imparting the enhanced agronomic trait. Homologs include proteins with an amino acid sequence that has at least 90% identity to a consensus amino acid sequence of proteins and homologs disclosed herein.

Homologs can be identified by comparison of amino acid sequence, e.g., manually or by using known homology-based search algorithms such as those commonly known and referred to as BLAST, FASTA, and Smith-Waterman. A local sequence alignment program, e.g., BLAST, can be used to search a database of sequences to find similar sequences, and the summary Expectation value (E-value) used to measure the sequence base similarity. As a protein hit with the best E-value for a particular organism may not necessarily be an ortholog or the only ortholog, a reciprocal query is used in the present invention to filter hit sequences with significant E-values for ortholog identification. The reciprocal query entails search of the significant hits against a database of amino acid sequences from the base organism that are similar to the sequence of the query protein. A hit is a likely ortholog, when the reciprocal query's best hit is the query protein itself or a protein encoded by a duplicated gene after speciation. A further aspect of the invention comprises functional homolog proteins which differ in one or more amino acids from those of disclosed protein as the result of conservative amino acid substitutions, e.g., substitutions are among: acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; basic (positively charged) amino acids such as arginine, histidine, and lysine; neutral polar amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; amino acids having aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; amino acids having aliphatic-hydroxyl side chains such as serine and threonine; amino acids having amide-containing side chains such as asparagine and glutamine; amino acids having aromatic side chains such as phenylalanine, tyrosine, and tryptophan; amino acids having basic side chains such as lysine, arginine, and histidine; amino acids having sulfur-containing side chains such as cysteine and methionine; naturally conservative amino acids such as valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. A further aspect of the homologs encoded by DNA useful in the transgenic plants of the invention are those proteins which differ from a disclosed protein as the result of deletion or insertion of one or more amino acids in a native sequence.

As used herein, “transcription factor gene” refers to a gene that encodes a protein that binds to regulatory regions and is involved in control gene expression. Therefore, as used herein, a target gene refers to a gene whose expression is controlled by a transcription factor gene.

As used herein, “percent identity” means the extent to which two optimally aligned DNA or protein segments are invariant throughout a window of alignment of components, e.g., nucleotide sequence or amino acid sequence. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by sequences of the two aligned segments divided by the total number of sequence components in the reference segment over a window of alignment which is the smaller of the full test sequence or the full reference sequence. “Percent identity” (“% identity”) is the identity fraction times 100.

As used herein “Pfam” refers to a large collection of multiple sequence alignments and hidden Markov models covering many common protein families, e.g. Pfam version 19.0 (December 2005) contains alignments and models for 8183 protein families and is based on the Swissprot 47.0 and SP-TrEMBL 30.0 protein sequence databases. See S. R. Eddy, “Profile Hidden Markov Models”, Bioinformatics 14:755-763, 1998. Pfam is currently maintained and updated by a Pfam Consortium. The alignments represent some evolutionary conserved structure that has implications for the protein's function. Profile hidden Markov models (profile HMMs) built from the Pfam alignments are useful for automatically recognizing that a new protein belongs to an existing protein family even if the homology by alignment appears to be low. Once one DNA is identified as encoding a protein which imparts an enhanced trait when expressed in transgenic plants, other DNA encoding proteins in the same protein family are identified by querying the amino acid sequence of protein encoded by candidate DNA against the Hidden Markov Model which characterizes the Pfam domain using HMMER software, a current version of which is provided in the appended computer listing. Candidate proteins meeting the gathering cutoff for the alignment of a particular Pfam are in the protein family and have cognate DNA that is useful in constructing recombinant DNA for the use in the plant cells of this invention. Hidden Markov Model databases for use with HMMER software in identifying DNA expressing protein in a common Pfam for recombinant DNA in the plant cells of this invention are also included in the appended computer listing. The HMMER software and Pfam databases are version 19.0 and were used to identify known domains in the proteins corresponding to amino acid sequence of SEQ ID NO: 194 through SEQ ID NO: 386. All DNA encoding proteins that have scores higher than the gathering cutoff disclosed in Table 11 by Pfam analysis disclosed herein can be used in recombinant DNA of the plant cells of this invention, e.g. for selecting transgenic plants having enhanced agronomic traits. The relevant Pfams for use in this invention, as more specifically disclosed below, are FAD_binding_(—)4, MtN3_slv, Homeobox, FAD_binding_(—)6, RWP-RK, PMEI, FAD_binding_(—)7, RRM_(—)1, Transaldolase, RNA_pol_L, WD40, U-box, Cyclin_N, Skp1, Redoxin, DZC, PBP, TPP_enzyme_M, CBFD_NFYB_HMF, TPP_enzyme_N, PFK, Caleosin, Iso_dh, Ribosomal_L18p, Metallophos, zf-A20, Ras, BBE, NAF, PLDc, DUF1242, Pkinase, C2, p450, Pyridoxal_deC, FBD, UPF0005, HEAT_PBS, GST_N, PEP-utilizers, Alpha-amylase, Amino_oxidase, SRF-TF, Phi_(—)1, Malic_M, Tryp_alpha_amyl, GSHPx, Miro, HSF_DNA-bind, DNA_photolyase, Sina, CTP_transf_(—)2, Abhydrolase_(—)3, Chal_sti_synt_C, ACP_syn_III_C, ADH_zinc_N, CSD, Globin, GATase_(—)2, Amidohydro_(—)1, HLH, HALZ, Amidohydro_(—)3, Lactamase_B, HSP20, DAO, DUF296, AT_hook, AWPM-19, Dimerisation, Suc_Fer-like, Methyltransf_(—)2, Aminotran_(—)3, PHD, MMR_HSR1, Aldo_ket_red, zf-AN1, malic, Fasciclin, UPF0057, DUF221, Pkinase_Tyr, DnaJ, Cofilin_ADF, Orn_Arg_deC_N, Skp1_POZ, Asn_synthase, K-box, LRR_(—)2, Ribosomal_L12, Ammonium_transp, Ribosomal_L14, KOW, DUF1336, DS, Aa_trans, CcmH, peroxidase, eIF-5a, Aldedh, PEP-utilizers_C, ADH_N, UIM, NAD_binding_(—)1, zf-C3HC4, Spermine_synth, AUX_IAA, LIM, Anti-silence, X8, Citrate_synt, 14-3-3, RMMBL, efhand, NPH3, CAF1, ICL, FAE1_CUT1_RppA, Orn_DAP_Arg_deC, PPDK_N, Myb_DNA-binding, AP2, F-box, and APS_kinase

As used herein, “promoter” means regulatory DNA for initializing transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell, e.g., is it well known that viral promoters are functional in plants. Thus, plant promoters include promoter DNA obtained from plants, plant viruses, and bacteria such as Agrobacterium and Rhizobium bacteria. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as “tissue preferred”. Promoters which initiate transcription only in certain tissues are referred to as “tissue specific”. A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” or “repressible” promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, or certain chemicals, or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter which is active under most conditions.

As used herein, “operably linked” means the association of two or more DNA fragments in a DNA construct so that the function of one, e.g., protein-encoding DNA, is affected by the other, e.g., a promoter.

As used herein, “expression” means the process that includes transcription of DNA to produce RNA and translation of the cognate protein encoded by the DNA and RNA.

As used herein, a “control plant” means a plant that does not contain the recombinant DNA that confers an enhanced agronomic trait. A control plant is used to compare against a transgenic plant, to identify an enhanced agronomic trait in the transgenic plant. A suitable control plant may be a non-transgenic plant of the parental line used to generate a transgenic plant. A control plant may in some cases be a transgenic plant line that comprises an empty vector or marker gene, but does not contain the recombinant DNA.

As used herein, an “agronomic trait” means a characteristic of a plant, which includes, but are not limited to, plant morphology, physiology, growth and development, yield, nutritional enhancement, disease or pest resistance, or environmental or chemical tolerance. In the plants of this invention the expression of identified recombinant DNA confers an agronomically important trait, e.g., increased yield. An “enhanced agronomic trait” refers to a measurable improvement in an agronomic trait including, but not limited to, yield increase, including increased yield under non-stress conditions and increased yield under environmental stress conditions. Stress conditions may include, for example, drought, shade, fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, cold temperature exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability and high plant density. “Yield” can be affected by many properties including without limitation, plant height, pod number, pod position on the plant, number of internodes, incidence of pod shatter, grain size, efficiency of nodulation and nitrogen fixation, efficiency of nutrient assimilation, resistance to biotic and abiotic stress, carbon assimilation, plant architecture, resistance to lodging, percent seed germination, seedling vigor, and juvenile traits. Yield can also affected by efficiency of germination (including germination in stressed conditions), growth rate (including growth rate in stressed conditions), ear number, seed number per ear, seed size, composition of seed (starch, oil, protein) and characteristics of seed fill.

Increased yield of a transgenic plant of the present invention can be measured in a number of ways, including test weight, seed number per plant, seed weight, seed number per unit area (i.e. seeds, or weight of seeds, per acre), bushels per acre, tones per acre, tons per acre, kilo per hectare. For example, maize yield may be measured as production of shelled corn kernels per unit of production area, e.g., in bushels per acre or metric tons per hectare, often reported on a moisture adjusted basis, e.g., at 15.5% moisture. Increased yield may result from enhanced utilization of key biochemical compounds, such as nitrogen, phosphorous and carbohydrate, or from improved responses to environmental stresses, such as cold, heat, drought, salt, and attack by pests or pathogens. Recombinant DNA used in this invention can also be used to provide plants having enhanced growth and development, and ultimately increased yield, as the result of modified expression of plant growth regulators or modification of cell cycle or photosynthesis pathways.

Also of interest is the generation of transgenic plants that demonstrate enhanced yield with respect to a seed component that may or may not correspond to an increase in overall plant yield. Such properties include enhancements in seed oil, seed molecules such as tocopherol, protein and starch, or oil particular oil components as may be manifest by an alteration in the ratios of seed components.

A subset of the nucleic molecules of this invention includes fragments of the disclosed recombinant DNA consisting of oligonucleotides of at least 15, preferably at least 16 or 17, more preferably at least 18 or 19, and even more preferably at least 20 or more, consecutive nucleotides. Such oligonucleotides are fragments of the larger molecules having a sequence selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:193, and find use, for example as probes and primers for detection of the polynucleotides of the present invention.

In some embodiments of the invention a constitutively active mutant is constructed to achieve the desired effect. SEQ ID NO: 3-6 encodes only the kinase domain of a calcium dependent protein kinase (CDPK). CDPK1 has a domain structure similar to other calcium-dependant protein kinase in which the protein kinase domain is separated from four efhand domains by 42 amino acid “spacer” region. Calcium-dependent protein kinases are thought to be activated by a calcium-induced conformational change that results in movement of an autoinhibitory domain away form the protein kinase active site (Yokokura et al., 1995). Thus, constitutively active proteins can be made by over expressing the protein kinase domain alone.

In other embodiments of the invention a chimeric gene is constructed between homologous genes from different species to obtain a protein with certain characteristics superior to either native protein, e.g., enhanced stability and favorable enzymatic kinetics. Exemplary chimeric DNA molecules provided by the present invention are set forth as SEQ ID NO: 1 and 2 that encode a Arabidopsis-Corn chimeric pyruvate orthophosphate dikinase (PPDK).

In yet other embodiments of the invention, a codon optimized gene is synthesized to achieve a desirable expression level. Synthetic DNA molecules can be designed by a variety of methods, such as, methods known in the art that are based upon substituting the codon(s) of a first polynucleotide to create an equivalent, or even an improved, second-generation artificial polynucleotide, where this new artificial polynucleotide is useful for enhanced expression in transgenic plants. The design aspect often employs a codon usage table. The table is produced by compiling the frequency of occurrence of codons in a collection of coding sequences isolated from a plant, plant type, family or genus. Other design aspects include reducing the occurrence of polyadenylation signals, intron splice sites, or long AT or GC stretches of sequence (U.S. Pat. No. 5,500,365). Full length coding sequences or fragments thereof can be made of artificial DNA using methods known to those skilled in the art. Such exemplary synthetic DNA molecules provided by the present invention are set forth as SEQ ID NO: 38.

DNA constructs are assembled using methods well known to persons of ordinary skill in the art and typically comprise a promoter operably linked to DNA, the expression of which provides the enhanced agronomic trait. Other construct components may include additional regulatory elements, such as 5′ introns for enhancing transcription, 3′ untranslated regions (such as polyadenylation signals and sites), DNA for transit or signal peptides.

In accordance with the current invention, constitutive promoters are active under most environmental conditions and states of development or cell differentiation. These promoters are likely to provide expression of the polynucleotide sequence at many stages of plant development and in a majority of tissues. A variety of constitutive promoters are known in the art. Examples of constitutive promoters that are active in plant cells include but are not limited to the nopaline synthase (NOS) promoters; the cauliflower mosaic virus (CaMV) 19S and 35S promoters (U.S. Pat. No. 5,858,642); the figwort mosaic virus promoter (P-FMV, U.S. Pat. No. 6,051,753); actin promoters, such as the rice actin promoter (P-Os.Act1, U.S. Pat. No. 5,641,876).

Furthermore, the promoters may be altered to contain one or more “enhancer sequences” to assist in elevating gene expression. Such enhancers are known in the art. By including an enhancer sequence with such constructs, the expression of the selected protein may be enhanced. These enhancers often are found 5′ to the start of transcription in a promoter that functions in eukaryotic cells, but can often be inserted in the forward or reverse orientation 5′ or 3′ to the coding sequence. In some instances, these 5′ enhancing elements are introns. Deemed to be particularly useful as enhancers are the 5′ introns of the rice actin 1 (see U.S. Pat. No. 5,641,876), rice actin 2 genes and the maize heat shock protein 70 gene intron (U.S. Pat. No. 5,593,874). Examples of other enhancers that can be used in accordance with the invention include elements from the CaMV 35S promoter, octopine synthase genes, the maize alcohol dehydrogenase gene, the maize shrunken 1 gene and promoters from non-plant eukaryotes.

Tissue-specific promoters cause transcription or enhanced transcription of a polynucleotide sequence in specific cells or tissues at specific times during plant development, such as in vegetative or reproductive tissues. Examples of tissue-specific promoters under developmental control include promoters that initiate transcription primarily in certain tissues, such as vegetative tissues, e.g., roots, leaves or stems, or reproductive tissues, such as fruit, ovules, seeds, pollen, pistils, flowers, or any embryonic tissue, or any combination thereof. Reproductive tissue specific promoters may be, e.g., ovule-specific, embryo-specific, endosperm-specific, integument-specific, pollen-specific, petal-specific, sepal-specific, or some combination thereof. Tissue specific promoter(s) will also include promoters that can cause transcription, or enhanced transcription in a desired plant tissue at a desired plant developmental stage. An example of such a promoter includes, but is not limited to, a seedling or an early seedling specific promoter. One skilled in the art will recognize that a tissue-specific promoter may drive expression of operably linked polynucleotide molecules in tissues other than the target tissue. Thus, as used herein, a tissue-specific promoter is one that drives expression preferentially not only in the target tissue, but may also lead to some expression in other tissues as well.

In one embodiment of this invention, preferential expression in plant green tissues is desired. Promoters of interest for such uses include those from genes such as maize aldolase gene FDA (U.S. patent application publication No. 20040216189), aldolase and pyruvate orthophosphate dikinase (PPDK) (Taniguchi et al. (2000) Plant Cell Physiol. 41(1):42-48).

In another embodiment of this invention, preferential expression in plant root tissue is desired. An exemplary promoter of interest for such uses is derived from Corn Nicotianamine Synthase gene (U.S. patent application publication No. 20030131377).

In yet another embodiment of this invention, preferential expression in plant phloem tissue is desired. An exemplary promoter of interest for such use is the rice tungro bacilliform virus (RTBV) promoter (U.S. Pat. No. 5,824,857).

In practicing this invention, an inducible promoter may also be used to ectopically express the structural gene in the recombinant DNA construct. The inducible promoter may cause conditional expression of a polynucleotide sequence under the influence of changing environmental conditions or developmental conditions. For example, such promoters may cause expression of the polynucleotide sequence at certain temperatures or temperature ranges, or in specific stage(s) of plant development such as in early germination or late maturation stage(s) of a plant. Examples of inducible promoters include, but are not limited to, the light-inducible promoter from the small subunit of ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO) (Fischhoff et al. (1992) Plant Mol. Biol. 20:81-93); the drought-inducible promoter of maize (Busk et al., Plant J. 11:1285-1295, 1997), the cold, drought, and high salt inducible promoter from potato (Kirch, Plant Mol. Biol. 33:897-909, 1997), and many cold inducible promoters known in the art; for example rd29a and cor15a promoters from Arabidopsis (Genbank ID: D13044 and U01377), blt101 and blt4.8 from barley (Genbank ID: AJ310994 and U63993), wcs120 from wheat (Genbank ID:AF031235), mlip15 from corn (Genbank ID: D26563) and bn115 from Brassica (Genbank ID: U01377).

In some aspects of the invention, sufficient expression in plant seed tissues is desired to effect improvements in seed composition. Exemplary promoters for use for seed composition modification include promoters from seed genes such as napin (U.S. Pat. No. 5,420,034), maize L3 oleosin (U.S. Pat. No. 6,433,252), zein Z27 (Russell et al. (1997) Transgenic Res. 6(2): 157-166), glutelin1 (Russell (1997) supra), peroxiredoxin antioxidant (Per1) (Stacy et al. (1996) Plant Mol. Biol. 31(6):1205-1216), and globulin 1 (Belanger et al (1991) Genetics 129:863-872).

Recombinant DNA constructs prepared in accordance with the invention will also generally include a 3′ element that typically contains a polyadenylation signal and site. Well-known 3′ elements include those from Agrobacterium tumefaciens genes such as nos 3′, tml 3′, tmr 3′, tms 3′, ocs 3′, tr7 3′, e.g., disclosed in U.S. Pat. No. 6,090,627, incorporated herein by reference; 3′ elements from plant genes such as wheat (Triticum aesevitum) heat shock protein 17 (Hsp173′), a wheat ubiquitin gene, a wheat fructose-1,6-biphosphatase gene, a rice glutelin gene a rice lactate dehydrogenase gene and a rice beta-tubulin gene, all of which are disclosed in U.S. published patent application 2002/0192813 A1, incorporated herein by reference; and the pea (Pisum sativum) ribulose biphosphate carboxylase gene (rbs 3′), and 3′ elements from the genes within the host plant.

Constructs and vectors may also include a transit peptide for targeting of a gene target to a plant organelle, particularly to a chloroplast, leucoplast or other plastid organelle. For descriptions of the use of chloroplast transit peptides see U.S. Pat. No. 5,188,642 and U.S. Pat. No. 5,728,925, incorporated herein by reference. For description of the transit peptide region of an Arabidopsis EPSPS gene useful in the present invention, see Klee, H. J. et al., (MGG (1987) 210:437-442).

The recombinant DNA construct may include other elements. For example, the construct may contain DNA segments that provide replication function and antibiotic selection in bacterial cells. For example, the construct may contain an E. coli origin of replication such as ori322 or a broad host range origin of replication such as oriV, oriRi or oriColE.

The construct may also comprise a selectable marker such as an Ec-ntpII-Tn5 that encodes a neomycin phosphotransferase II gene obtained from Tn5 conferring resistance to a neomycin and kanamysin, Spc/Str that encodes for Tn7 aminoglycoside adenyltransferase (aadA) conferring resistance to spectinomycin or streptomycin, or a gentamicin (Gm, Gent) or one of many known selectable marker gene.

The vector or construct may also include a screenable marker and other elements as appropriate for selection of plant or bacterial cells having DNA constructs of the invention. DNA constructs are designed with suitable selectable markers that can confer antibiotic or herbicide tolerance to the cell. The antibiotic tolerance polynucleotide sequences include, but are not limited to, polynucleotide sequences encoding for proteins involved in tolerance to kanamycin, neomycin, hygromycin, and other antibiotics known in the art. An antibiotic tolerance gene in such a vector may be replaced by herbicide tolerance gene encoding for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, described in U.S. Pat. Nos. 5,627,061, and 5,633,435; Padgette et al., Herbicide Resistant Crops, Lewis Publishers, 53-85, 1996; and in Penaloza-Vazquez, et al., Plant Cell Reports 14:482-487, 1995) and aroA (U.S. Pat. No. 5,094,945) for glyphosate tolerance, bromoxynil nitrilase (Bxn) for Bromoxynil tolerance (U.S. Pat. No. 4,810,648), phytoene desaturase (crtI (Misawa et al., Plant J. 4:833-840, 1993; and Misawa et al., Plant J. 6:481-489, 1994) for tolerance to norflurazon, acetohydroxyacid synthase (AHAS, Sathasiivan et al., Nucl. Acids Res. 18:2188-2193, 1990). Herbicides for which transgenic plant tolerance has been demonstrated and for which the method of the present invention can be applied include, but are not limited to: glyphosate, sulfonylureas, imidazolinones, bromoxynil, delapon, cyclohezanedione, protoporphyrionogen oxidase inhibitors, and isoxaslutole herbicides.

Other examples of selectable markers, screenable markers and other elements are well known in the art and may be readily used in the present invention. Those skilled in the art should refer to the following for details (for selectable markers, see Potrykus et al., Mol. Gen. Genet. 199:183-188, 1985; Hinchee et al., Bio. Techno. 6:915-922, 1988; Stalker et al., J. Biol. Chem. 263:6310-6314, 1988; European Patent Application 154,204; Thillet et al., J. Biol. Chem. 263:12500-12508, 1988; for screenable markers see, Jefferson, Plant Mol. Biol, Rep. 5: 387-405, 1987; Jefferson et al., EMBO J. 6: 3901-3907, 1987; Sutcliffe et al., Proc. Natl. Acad. Sci. U.S.A. 75: 3737-3741, 1978; Ow et al., Science 234: 856-859, 1986; Ikatu et al., Bio. Technol. 8: 241-242, 1990; and for other elements see, European Patent Application Publication Number 0218571; Koziel et al., Plant Mol. Biol. 32: 393-405; 1996).

The plants of this invention can be further enhanced with stacked traits, e.g., a crop having an enhanced agronomic trait resulting from expression of DNA disclosed herein, in combination with herbicide, disease, and/or pest resistance traits. The recombinant DNA is provided in plant cells derived from corn lines that maintain resistance to a virus such as the Mal de Rio Cuarto virus or a fungus such as the Puccina sorghi fungus or both, which are common plant diseases in Argentina. For example, genes of the current invention can be stacked with other traits of agronomic interest, such as a trait providing herbicide resistance, or insect resistance, such as using a gene from Bacillus thuringiensis to provide resistance against lepidopteran, coleopteran, homopteran, hemiopteran, and other insects. Herbicides for which transgenic plant tolerance has been demonstrated and the method of the present invention can be applied include, but are not limited to, glyphosate, dicamba, glufosinate, sulfonylurea, bromoxynil and norflurazon herbicides. Polynucleotide molecules encoding proteins involved in herbicide tolerance are well-known in the art and include, but are not limited to, a polynucleotide molecule encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) disclosed in U.S. Pat. Nos. 5,094,945; 5,627,061; 5,633,435 and 6,040,497 for imparting glyphosate tolerance; polynucleotide molecules encoding a glyphosate oxidoreductase (GOX) disclosed in U.S. Pat. No. 5,463,175 and a glyphosate-N-acetyl transferase (GAT) disclosed in U.S. Patent Application publication 2003/0083480 A1 also for imparting glyphosate tolerance; dicamba monooxygenase disclosed in U.S. Patent Application publication 2003/0135879 A1 for imparting dicamba tolerance; a polynucleotide molecule encoding bromoxynil nitrilase (Bxn) disclosed in U.S. Pat. No. 4,810,648 for imparting bromoxynil tolerance; a polynucleotide molecule encoding phytoene desaturase (crtI) described in Misawa et al, (1993) Plant J. 4:833-840 and Misawa et al, (1994) Plant J. 6:481-489 for norflurazon tolerance; a polynucleotide molecule encoding acetohydroxyacid synthase (AHAS, aka ALS) described in Sathasiivan et al. (1990) Nucl. Acids Res. 18:2188-2193 for imparting tolerance to sulfonylurea herbicides; polynucleotide molecules known as bar genes disclosed in DeBlock, et al. (1987) EMBO J. 6:2513-2519 for imparting glufosinate and bialaphos tolerance; polynucleotide molecules disclosed in U.S. Patent Application Publication 2003/010609 A1 for imparting N-amino methyl phosphonic acid tolerance; polynucleotide molecules disclosed in U.S. Pat. No. 6,107,549 for imparting pyridine herbicide resistance; molecules and methods for imparting tolerance to multiple herbicides such as glyphosate, atrazine, ALS inhibitors, isoxoflutole and glufosinate herbicides are disclosed in U.S. Pat. No. 6,376,754 and U.S. Patent Application Publication 2002/0112260, all of said U.S. patents and patent application publications are incorporated herein by reference. Molecules and methods for imparting insect/nematode/virus resistance is disclosed in U.S. Pat. Nos. 5,250,515; 5,880,275; 6,506,599; 5,986,175 and U.S. Patent Application Publication 2003/0150017 A1, all of which are incorporated herein by reference.

In particular embodiments, the inventors contemplate the use of antibodies, either monoclonal or polyclonal which bind to the proteins disclosed herein. Means for preparing and characterizing antibodies are well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference). The methods for generating monoclonal antibodies (mAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.

As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include using glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.

As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.

The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and tittering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.

mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified antifungal protein, polypeptide or peptide. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep, or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).

Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986, pp. 65-66; Campbell, 1984, pp. 75-83). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XXO Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (also termed P3-NS-1-Ag-4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.

Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Spend virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, (Gefter et al., 1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986, pp. 71-74).

Fusion procedures usually produce viable hybrids at low frequencies, about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azasenne blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.

This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.

The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.

Transformation Method

Numerous methods for transforming plant cells with recombinant DNA are known in the art and may be used in the present invention. Two commonly used methods for plant transformation are Agrobacterium-mediated transformation and microprojectile bombardment. Microprojectile bombardment methods are illustrated in U.S. Pat. Nos. 5,015,580 (soybean); 5,550,318 (corn); 5,538,880 (corn); 5,914,451 (soybean); 6,160,208 (corn); 6,399,861 (corn) and 6,153,812 (wheat) and Agrobacterium-mediated transformation is described in U.S. Pat. Nos. 5,159,135 (cotton); 5,824,877 (soybean); 5,591,616 (corn); and 6,384,301 (soybean), and in US Patent Application Publication 2004/0244075, all of which are incorporated herein by reference. For Agrobacterium tumefaciens based plant transformation system, additional elements present on transformation constructs will include T-DNA left and right border sequences to facilitate incorporation of the recombinant polynucleotide into the plant genome.

In general it is useful to introduce recombinant DNA randomly, i.e. at a non-specific location, in the genome of a target plant line. In special cases it may be useful to target recombinant DNA insertion in order to achieve site-specific integration, e.g., to replace an existing gene in the genome, to use an existing promoter in the plant genome, or to insert a recombinant polynucleotide at a predetermined site known to be active for gene expression. Several site specific recombination systems exist which are known to function implants include cre-lox as disclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S. Pat. No. 5,527,695, both incorporated herein by reference.

Transformation methods of this invention are preferably practiced in tissue culture on media and in a controlled environment. “Media” refers to the numerous nutrient mixtures that are used to grow cells in vitro, that is, outside of the intact living organism. Recipient cell targets include, but are not limited to, meristem cells, callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. It is contemplated that any cell from which a fertile plant may be regenerated is useful as a recipient cell. Callus may be initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, microspores and the like. Cells capable of proliferating as callus are also recipient cells for genetic transformation. Practical transformation methods and materials for making transgenic plants of this invention, e.g., various media and recipient target cells, transformation of immature embryos and subsequent regeneration of fertile transgenic plants are disclosed in U.S. Pat. Nos. 6,194,636 and 6,232,526, which are incorporated herein by reference.

The seeds of transgenic plants can be harvested from fertile transgenic plants and be used to grow progeny generations of transformed plants of this invention including hybrid plants line for screening of plants having an enhanced agronomic trait. In addition to direct transformation of a plant with a recombinant DNA, transgenic plants can be prepared by crossing a first plant having a recombinant DNA with a second plant lacking the DNA. For example, recombinant DNA can be introduced into first plant line that is amenable to transformation to produce a transgenic plant which can be crossed with a second plant line to introgress the recombinant DNA into the second plant line. A transgenic plant with recombinant DNA providing an enhanced agronomic trait, e.g., enhanced yield, can be crossed with transgenic plant line having other recombinant DNA that confers another trait, e.g., herbicide resistance or pest resistance, to produce progeny plants having recombinant DNA that confers both traits. Typically, in such breeding for combining traits the transgenic plant donating the additional trait is a male line and the transgenic plant carrying the base traits is the female line. The progeny of this cross will segregate such that some of the plants will carry the DNA for both parental traits and some will carry DNA for one parental trait; such plants can be identified by markers associated with parental recombinant DNA Progeny plants carrying DNA for both parental traits can be crossed back into the female parent line multiple times, e.g., usually 6 to 8 generations, to produce a progeny plant with substantially the same genotype as one original transgenic parental line but for the recombinant DNA of the other transgenic parental line.

In the practice of transformation DNA is typically introduced into only a small percentage of target cells in any one transformation experiment. Marker genes are used to provide an efficient system for identification of those cells that are stably transformed by receiving and integrating a transgenic DNA construct into their genomes. Preferred marker genes provide selective markers which confer resistance to a selective agent, such as an antibiotic or herbicide. Any of the herbicides to which plants of this invention may be resistant are useful agents for selective markers. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene is integrated and expressed at sufficient levels to permit cell survival. Cells may be tested further to confirm stable integration of the exogenous DNA. Commonly used selective marker genes include those conferring resistance to antibiotics such as kanamycin and paromomycin (nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat) and glyphosate (aroA or EPSPS). Examples of such selectable are illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047, all of which are incorporated herein by reference. Screenable markers which provide an ability to visually identify transformants can also be employed, e.g., a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a beta-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known.

Cells that survive exposure to the selective agent, or cells that have been scored positive in a screening assay, may be cultured in regeneration media and allowed to mature into plants. Developing plantlets can be transferred to plant growth mix, and hardened off, e.g., in an environmentally controlled chamber at about 85% relative humidity, 600 ppm CO₂, and 25-250 microeinsteins m⁻²s⁻¹ of light, prior to transfer to a greenhouse or growth chamber for maturation. Plants are regenerated from about 6 weeks to 10 months after a transformant is identified, depending on the initial tissue. Plants may be pollinated using conventional plant breeding methods known to those of skill in the art and seed produced, e.g., self-pollination is commonly used with transgenic corn. The regenerated transformed plant or its progeny seed or plants can be tested for expression of the recombinant DNA and screened for the presence of enhanced agronomic trait.

Transgenic Plants and Seeds

Transgenic plant seed provided by this invention are grown to generate transgenic plants having an enhanced trait as compared to a control plant. Such seed for plants with enhanced agronomic trait is identified by screening transformed plants or progeny seed for enhanced trait. For efficiency a screening program is designed to evaluate multiple transgenic plants (events) comprising the recombinant DNA, e.g., multiple plants from 2 to 20 or more transgenic events.

Transgenic plants grown from transgenic seed provided herein demonstrate enhanced agronomic traits that contribute to increased yield or other trait that provides increased plant value, including, for example, enhanced seed quality. Of particular interest are plants having enhanced yield resulting from enhanced plant growth and development, stress tolerance, enhanced seed development, higher light response, enhanced flower development, or enhanced carbon and/or nitrogen metabolism.

TABLE 1 NUC PEP annotation SEQ SEQ Base e- ID NO ID NO vector GENE ID value % identity identifier description 1 194 1 PHE0003351_PMON81242 0 98 168586 gb|AAA33498.1|pyruvate, orthophosphate dikinase 2 195 7 PHE0003351_PMON83625 0 98 168586 gb|AAA33498.1|pyruvate, orthophosphate dikinase 3 196 1 PHE0000207_PMON77878 1.00E−144 96 34907990 ref|NP_915342.1|putative calcium-dependent protein kinase [Oryza sativa (japonica cultivar-group)] 4 197 1 PHE0000208_PMON77879 1.00E−143 94 50919297 ref|XP_470045.1|putative calmodulin-domain protein kinase [Oryza sativa (japonica cultivar-group)] 5 198 1 PHE0000209_PMON77891 1.00E−135 89 53850561 gb|AAU95457.1|At5g12180 [Arabidopsis thaliana] dbj|BAB10036.1|calcium- dependent protein kinase 6 199 1 PHE0000210_PMON77880 1.00E−137 89 26452430 dbj|BAC43300.1|putative calcium-dependent protein kinase [Arabidopsis thaliana] 7 200 8 PHE0001329_PMON92878 0 100 34903780 dbj|BAB92151.1|putative CBL-interacting protein kinase 2 [Oryza sativa (japonica 8 201 1 PHE0001425_PMON79162 1.00E−154 100 51979679 ref|XP_507586.1|PREDICTED P0524F03.33 gene product [Oryza sativa (japonica cultivar-group)] ref|XP_482612.1|putative CCR4-NOT transcription complex, subunit 7 9 202 8 PHE0001573_PMON92870 0 78 984262 emb|CAA58052.1|asparragine synthetase [Zea mays] 10 203 12 PHE0001664_PMON99280 0 100 34906358 sp|Q9LDE6|CKX1_ORYS A Probable cytokinin dehydrogenase precursor (Cytokinin oxidase) (CKO) 11 204 1 PHE0001674_PMON79194 5.00E−12 50 15223390 ref|NP_171645.1|myb family transcription factor [Arabidopsis thaliana] 12 205 10 PHE0002026_PMON96489 0 87 32488298 emb|CAE03364.1|OSJNBb0065L13.7 [Oryza sativa (japonica cultivar-group)] 13 206 8 PHE0002108_PMON92821 2.00E−31 100 10176234 dbj|BAB07329.1|cold-shock protein [Bacillus halodurans C-125] 14 207 8 PHE0002109_PMON93856 6.00E−33 100 41324401 emb|CAF18741.1|COLD- SHOCK PROTEIN CSPA [Corynebacterium glutamicum ATCC 13032] 15 208 8 PHE0002508_PMON92607 2.00E−79 72 50509850 dbj|BAD32022.1|putative transcription factor [Oryza sativa 16 209 1 PHE0002650_PMON81832 1.00E−132 100 9964296 gb|AAG09919.1|MADS box protein 2 [Zea mays] 17 210 2 PHE0002989_PMON95630 1.00E−117 100 7271044 emb|CAB80652.1|small GTP-binding protein-like [Arabidopsis thaliana] 18 211 6 PHE0003290_PMON95107 4.00E−29 34 7269078 emb|CAB79187.1|hypothetical protein [Arabidopsis thaliana] 19 212 6 PHE0003300_PMON95106 7.00E−18 54 50908933 ref|XP_465955.1|putative nodulin 3 [Oryza sativa (japonica cultivar-group)] 20 213 6 PHE0003303_PMON95080 2.00E−96 69 38347194 emb|CAD37109.2|OSJNBa0024J22.22 [Oryza sativa (japonica cultivar-group)] 21 214 8 PHE0003389_PMON94682 0 65 52076827 dbj|BAD45770.1|putative Cyt-P450 monooxygenase [Oryza sativa (japonica cultivar-group)] 22 215 8 PHE0003614_PMON95111 0 94 32309578 gb|AAP79441.1|glutamate decarboxylase [Oryza sativa (japonica cultivar-group)] 23 216 8 PHE0003684_PMON92807 1.00E−72 68 34906004 dbj|BAB63676.1|induced protein MgI1 [Oryza sativa (japonica cultivar-group)] 24 217 9 PHE0003684_PMON93378 1.00E−72 68 34906004 dbj|BAB63676.1|induced protein MgI1 [Oryza sativa (japonica cultivar-group)] 25 218 8 PHE0003853_PMON92602 1.00E−179 98 62320210 ref|NP_195478.2|cyclin family protein [Arabidopsis thaliana] gb|AAS49095.1| At4g37630 [Arabidopsis thaliana] 26 219 11 PHE0003903_PMON98271 0 99 19851522 gb|AAL99744.1|pyruvate decarboxylase [Zea mays] 27 220 11 PHE0003905_PMON99283 0 92 11995457 gb|AAG43027.1|aldehyde dehydrogenase [Oryza sativa] 28 221 11 PHE0003907_PMON98066 5.00E−87 86 50906015 ref|XP_464496.1|ribosomal protein L12-like protein [Oryza sativa (japonica cultivar-group)] 29 222 11 PHE0003908_PMON98064 0 84 51535811 dbj|BAD37896.1|ARG1- like protein [Oryza sativa (japonica cultivar-group)] 30 223 6 PHE0003960_PMON95079 1.00E−156 87 50905641 ref|XP_464309.1|putative choline-phosphate cytidylyltransferase [Oryza sativa (japonica cultivar- group)] 31 224 5 PHE0003967_PMON95088 1.00E−102 83 55168334 gb|AAV44199.1|dehydroascorbate reductase [Oryza sativa (japonica cultivar- group)] 32 225 10 PHE0003985_PMON96457 1.00E−30 58 55770043 ref|XP_550011.1|hypothetical protein [Oryza sativa (japonica cultivar-group)] 33 226 10 PHE0003987_PMON96134 5.00E−41 74 50919885 ref|XP_470303.1|hypothetical protein [Oryza sativa (japonica cultivar-group)] 34 227 10 PHE0004001_PMON96453 4.00E−22 66 51978970 ref|XP_507362.1|PREDICTED OSJNBa0077F02.127 gene product [Oryza sativa (japonica cultivar-group)] 35 228 8 PHE0004023_PMON92446 1.00E−132 88 12651665 gb|AAA20093.2|Alfin-1 [Medicago sativa] pir||T09646 probable zinc finger protein - alfalfa (fragment) 36 229 4 PHE0004026_PMON93885 0 100 21592703 gb|AAM64652.1|LAX1/ AUX1-like permease [Arabidopsis thaliana] 37 230 4 PHE0004027_PMON93860 0 100 7269873 emb|CAB79732.1|cytokinin oxidase-like protein [Arabidopsis thaliana] 38 231 15 PHE0004028_PMON94697 0 100 216765 dbj|BAA14344.1|sucrose phosphorylase [Leuconostoc mesenteroides] 12607 231 n/a PHE0010424_PMON17730 0 100 216765 dbj|BAA14344.1|sucrose phosphorylase [Leuconostoc mesenteroides] 39 232 8 PHE0004034_PMON92631 0 100 6520233 dbj|BAA87958.1|CW14 [Arabidopsis thaliana] 40 233 8 PHE0004039_PMON92634 1.00E−178 65 26452061 ref|NP_191207.2|myosin heavy chain-related [Arabidopsis thaliana] 41 234 8 PHE0004047_PMON92619 4.00E−79 74 62087121 dbj|BAD91881.1|transcription factor lim1 [Eucalyptus camaldulensis] 42 235 14 PHE0004047_PMON93388 4.00E−79 74 62087121 dbj|BAD91881.1|transcription factor lim1 [Eucalyptus camaldulensis] 43 236 8 PHE0004068_PMON93663 3.00E−94 100 15293293 ref|NP_563710.1|AWPM- 19-like membrane family protein [Arabidopsis thaliana] 44 237 8 PHE0004071_PMON93311 1.00E−130 100 21358850 ref|NP_568751.1| polyadenylate-binding protein, putative/PABP, putative [Arabidopsis thaliana] 45 238 8 PHE0004072_PMON93654 0 100 23297397 ref|NP_192188.2|GTP- binding family protein [Arabidopsis thaliana] 46 239 14 PHE0004072_PMON93669 0 100 23297397 ref|NP_192188.2|GTP- binding family protein [Arabidopsis thaliana] 47 240 8 PHE0004074_PMON94164 0 100 9759255 ref|NP_196133.3| transcription elongation factor-related [Arabidopsis thaliana] 48 241 8 PHE0004075_PMON92851 1.00E−132 100 11994587 ref|NP_566493.1|nodulin MtN3 family protein [Arabidopsis thaliana] 49 242 8 PHE0004080_PMON93321 1.00E−143 99 16173 emb|CAA42168.1|L- ascorbate peroxidase [Arabidopsis thaliana] 50 243 14 PHE0004084_PMON95141 0 100 7267537 emb|CAB78019.1|putative phi-1-like phosphate- induced protein [Arabidopsis thaliana] gb|AAM18526.1| cell cycle- related protein [Arabidopsis thaliana] 51 244 8 PHE0004093_PMON93332 0 100 12744973 gb|AAK06866.1|putative ATPase [Arabidopsis thaliana] ref|NP_173536.1| O-methyltransferase, putative [Arabidopsis thaliana] 52 245 14 PHE0004093_PMON94155 0 100 12744973 gb|AAK06866.1|putative ATPase [Arabidopsis thaliana] ref|NP_173536.1| O-methyltransferase, putative [Arabidopsis thaliana] 53 246 8 PHE0004139_PMON92898 2.00E−88 100 21554099 ref|NP_568761.1| expressed protein [Arabidopsis thaliana] 54 247 8 PHE0004144_PMON93842 1.00E−78 100 21555039 ref|NP_565390.1| actin- depolymerizing factor 5 (ADF5) [Arabidopsis thaliana] 55 248 8 PHE0004148_PMON92574 0 100 48768596 ref|ZP_00272945.1|COG0538: Isocitrate dehydrogenases [Ralstonia metallidurans CH34] 56 249 8 PHE0004149_PMON92471 1.00E−148 99 31096331 ref|NP_441003.1| phycocyanin alpha phycocyanobilin lyase; CpcE [Synechocystis sp. PCC 6803] 57 250 14 PHE0004149_PMON93899 1.00E−148 99 31096331 ref|NP_441003.1| phycocyanin alpha phycocyanobilin lyase; CpcE [Synechocystis sp. PCC 6803] 58 251 15 PHE0004152_PMON93672 3.00E−85 60 8978267 ref|NP_199781.1|DNA- binding protein-related [Arabidopsis thaliana] 59 252 8 PHE0004155_PMON92626 0 100 22136876 ref|NP_200010.1|sorbitol dehydrogenase, putative/ L-iditol 2-dehydrogenase, putative [Arabidopsis thaliana] 60 253 8 PHE0004156_PMON92623 0 98 12322729 ref|NP_187478.1| phototropic-responsive protein, putative [Arabidopsis thaliana] 61 254 8 PHE0004162_PMON92481 3.00E−77 57 7269806 emb|CAB79666.1|phytochrome- associated protein PAP2 [Arabidopsis thaliana] 62 255 8 PHE0004164_PMON92465 4.00E−67 100 21537028 ref|NP_198423.1|glycosyl hydrolase family protein 17 [Arabidopsis thaliana] 63 256 8 PHE0004166_PMON93801 6.00E−09 100 13374861 emb|CAC34495.1|putative strictosidine synthase-like [Arabidopsis thaliana] 64 257 8 PHE0004167_PMON93333 1.00E−176 100 28827764 ref|NP_569050.1| adenylylsulfate kinase, putative [Arabidopsis thaliana] 65 258 8 PHE0004168_PMON93855 0 100 18176302 ref|NP_199253.1|FAD- binding domain-containing protein [Arabidopsis thaliana] 66 259 8 PHE0004169_PMON92568 0 100 5080826 gb|AAD39335.1|Putative Aldo/keto reductase [Arabidopsis thaliana] 67 260 8 PHE0004184_PMON92565 0 100 7270846 emb|CAB80527.1|multiubiquitin chain binding protein (MBP1) [Arabidopsis thaliana] 68 261 8 PHE0004185_PMON92802 0 100 28460683 ref|NP_182075.1| cytochrome P450, putative [Arabidopsis thaliana] 69 262 8 PHE0004188_PMON92803 0 100 20465485 ref|NP_200218.1|heat shock transcription factor family protein [Arabidopsis thaliana] 70 263 8 PHE0004190_PMON92801 1.00E−167 98 7267277 ref|NP_192426.1|basic helix-loop-helix (bHLH) family protein [Arabidopsis thaliana] 71 264 8 PHE0004208_PMON92834 1.00E−83 55 21555865 gb|AAS09998.1|MYB transcription factor [Arabidopsis thaliana] 72 265 8 PHE0004215_PMON92827 2.00E−55 65 7320708 ref|NP_195750.1| phosphatidylethanolamine- binding family protein [Arabidopsis thaliana] 73 266 8 PHE0004223_PMON92840 0 100 6523058 ref|NP_190239.1|fasciclin- like arabinogalactan family protein [Arabidopsis thaliana] 74 267 8 PHE0004225_PMON94167 0 99 1421730 gb|AAC49371.1|RF2 gb|AAG43988.1|T cytoplasm male sterility restorer factor 2 [Zea mays] 75 268 10 PHE0004226_PMON95114 0 100 53793208 dbj|BAD54414.1|aldehyde dehydrogenase ALDH2b [Oryza sativa (japonica cultivar-group)] 76 269 8 PHE0004227_PMON92605 5.00E−26 100 21314334 gb|AAM46894.1|early drought induced protein [Oryza sativa (indica cultivar-group)] 77 270 8 PHE0004229_PMON92867 1.00E−24 100 6320482 ref|NP_010562.1|Small plasma membrane protein related to a family of plant polypeptides that are overexpressed under high salt concentration or low temperature, not essential for viability, deletion causes hyperpolarization of the plasma membrane potential; Pmp3p [Saccharomyces cerevisiae] 78 271 8 PHE0004233_PMON92843 0 100 19310749 ref|NP_188922.1|heat shock transcription factor family protein [Arabidopsis thaliana] 79 272 13 PHE0004237_PMON93673 9.00E−85 100 16338 emb|CAA45039.1|heat shock protein 17.6-II [Arabidopsis thaliana] 80 273 8 PHE0004243_PMON92621 3.00E−72 82 30409461 dbj|BAC76332.1|HAP3 [Oryza sativa (japonica cultivar-group)] 81 274 8 PHE0004244_PMON92858 1.00E−159 96 15321716 gb|AAK95562.1|leafy cotyledon1 [Zea mays] 82 275 8 PHE0004245_PMON93813 1.00E−131 100 50509850 dbj|BAD32022.1|putative transcription factor [Oryza sativa (japonica cultivar- group)] 83 276 8 PHE0004248_PMON94672 1.00E−98 100 34907184 ref|NP_914939.1|putative CCAAT-binding transcription factor subunit A(CBF-A) [Oryza sativa 84 277 8 PHE0004249_PMON95137 1.00E−48 100 12642910 ref|NP_850005.1|expressed protein [Arabidopsis thaliana] 85 278 8 PHE0004250_PMON92881 5.00E−78 100 30409463 dbj|BAC76333.1|HAP3 [Oryza sativa (japonica cultivar-group)] 86 279 8 PHE0004252_PMON92606 1.00E−173 100 18481620 gb|AAL73485.1|repressor protein [Oryza sativa] 87 280 8 PHE0004253_PMON92874 1.00E−143 100 18481626 gb|AAL73488.1|repressor protein [Zea mays] 88 281 14 PHE0004258_PMON93385 0 100 1871189 gb|AAB63549.1|putative protein kinase [Arabidopsis thaliana] 89 282 8 PHE0004258_PMON93806 0 100 1871189 gb|AAB63549.1|putative protein kinase [Arabidopsis thaliana] 90 283 14 PHE0004259_PMON93384 0 100 9755654 ref|NP_197112.1|expressed protein [Arabidopsis thaliana] 91 284 8 PHE0004260_PMON92854 1.00E−48 100 12642910 ref|NP_850005.1|expressed protein [Arabidopsis thaliana] 92 285 14 PHE0004261_PMON93389 1.00E−170 100 7270230 ref|NP_195009.1|protein kinase, putative [Arabidopsis thaliana] 93 286 8 PHE0004261_PMON93655 1.00E−170 100 7270230 ref|NP_195009.1|protein kinase, putative [Arabidopsis thaliana] 94 287 8 PHE0004262_PMON92862 0 100 42570809 ref|NP_973478.1|protein kinase, putative [Arabidopsis thaliana] 95 288 14 PHE0004262_PMON93360 0 100 42570809 ref|NP_973478.1|protein kinase, putative [Arabidopsis thaliana] 96 289 8 PHE0004264_PMON92845 3.00E−95 100 21554624 ref|NP_201267.1| invertase/pectin methylesterase inhibitor family protein [Arabidopsis thaliana] 97 290 14 PHE0004264_PMON93354 3.00E−95 100 21554624 ref|NP_201267.1| invertase/pectin methylesterase inhibitor family protein [Arabidopsis thaliana] 98 291 8 PHE0004265_PMON92873 0 100 642305 ref|NP_013662.1| Hypothetical ORF; Yml050wp [Saccharomyces cerevisiae] 99 292 14 PHE0004265_PMON93807 0 100 642305 ref|NP_013662.1| Hypothetical ORF; Yml050wp [Saccharomyces cerevisiae] 100 293 8 PHE0004266_PMON92877 0 99 23506085 ref|NP_567548.1|pseudo- response regulator 2 (APRR2) (TOC2) [Arabidopsis thaliana] 101 294 8 PHE0004284_PMON93857 0 99 18399375 ref|NP_566402.1|U-box domain-containing protein [Arabidopsis thaliana] 102 295 10 PHE0004285_PMON95136 1.00E−161 96 37542675 gb|AAL47207.1|HAP3-like transcriptional-activator [Oryza sativa (indica cultivar-group)] 103 296 8 PHE0004286_PMON93666 0 99 255220 gb|AAB23208.1|isocitrate lyase, threo-D S-isocitrate glyoxylate-lyase, IL {EC 4.1.3.1} [Brassica napus, seedlings, Peptide, 576 aa] 104 297 8 PHE0004287_PMON93344 0 88 50937953 ref|XP_478504.1|putative isocitrate lyase [Oryza sativa (japonica cultivar- group)] 105 298 2 PHE0004307_PMON94102 1.00E−105 62 38345397 emb|CAE03088.2|OSJNBa0017B10.3 [Oryza sativa (japonica cultivar-group)] 106 299 14 PHE0004314_PMON93397 9.00E−52 54 55740645 gb|AAV63915.1|hypothetical protein At4g03965 [Arabidopsis thaliana] 107 300 8 PHE0004321_PMON93811 1.00E−128 100 18655355 sp|O48646|GPX4_ARATH Probable phospholipid hydroperoxide glutathione peroxidase, mitochondrial precursor (PHGPx) (AtGPX1) 108 301 14 PHE0004321_PMON93834 1.00E−128 100 18655355 ref|NP_192897.2| glutathione peroxidase, putative [Arabidopsis thaliana] 109 302 8 PHE0004325_PMON93818 5.00E−78 89 50906887 ref|XP_464932.1|cytochrome c biogenesis protein-like [Oryza sativa (japonica cultivar-group)] 110 303 8 PHE0004335_PMON93850 0 100 28393953 gb|AAO42384.1|putative major intrinsic protein [Arabidopsis thaliana] 111 304 8 PHE0004336_PMON93858 1.00E−146 69 51964952 ref|XP_482812.1|major intrinsic protein-like [Oryza sativa (japonica cultivar- group)] 112 305 4 PHE0004337_PMON93886 0 62 50943587 ref|XP_481321.1|unknown protein [Oryza sativa (japonica cultivar-group)] 113 306 8 PHE0004348_PMON93810 1.00E−32 100 15644431 ref|NP_229483.1|cold shock protein [Thermotoga maritima MSB8] 114 307 8 PHE0004349_PMON93812 8.00E−33 100 15644617 ref|NP_229670.1|cold shock protein [Thermotoga maritima MSB8] 115 308 8 PHE0004350_PMON93826 3.00E−31 100 20808157 ref|NP_623328.1|Cold shock proteins [Thermoanaerobacter tengcongensis MB4] 116 309 8 PHE0004351_PMON93821 7.00E−32 100 56419891 ref|YP_147209.1|cold shock protein [Geobacillus kaustophilus HTA426] 117 310 8 PHE0004352_PMON93824 1.00E−27 88 49611845 ref|YP_050486.1|cold shock protein [Erwinia carotovora subsp. atroseptica SCRI1043] 118 311 8 PHE0004383_PMON93816 1.00E−34 98 50899510 ref|XP_450543.1|unknown protein [Oryza sativa (japonica cultivar-group)] 119 312 8 PHE0004393_PMON94192 8.00E−95 100 42572939 ref|NP_974566.1|calcineurin B-like protein 1 (CBL1) [Arabidopsis thaliana] 120 313 8 PHE0004395_PMON94145 0 100 30690488 ref|NP_849501.1|phospholipase D delta/PLD delta (PLDDELTA) [Arabidopsis thaliana] 121 314 8 PHE0004396_PMON94137 0 100 7270422 emb|CAB80188.1|arginine decarboxylase SPE2 [Arabidopsis thaliana] 122 315 8 PHE0004417_PMON94190 1.00E−170 100 1230677 gb|AAC17191.1| spermidine synthase [Saccharomyces cerevisiae] 123 316 8 PHE0004418_PMON94368 0 100 798930 sp|P50264|FMS1_YEAST Polyamine oxidase FMS1 (Fenpropimorph resistance multicopy suppressor 1) 124 317 8 PHE0004419_PMON95100 0 66 21281139 ref|NP_567276.1| amidohydrolase family protein [Arabidopsis thaliana] 125 318 10 PHE0004421_PMON95120 2.00E−53 78 33321848 gb|AAQ06658.1|apetala2 domain-containing CBF1- like protein [Oryza sativa] 126 319 10 PHE0004422_PMON95123 3.00E−51 80 25991254 gb|AAN76804.1|DREB-like protein [Zea mays] 127 320 8 PHE0004425_PMON94428 7.00E−37 98 11762134 gb|AAG40345.1|AT5g17460 [Arabidopsis thaliana] 128 321 8 PHE0004431_PMON94398 1.00E−159 99 557818 ref|NP_012214.1|Pho85p cyclin of the Pho80p subfamily, forms a functional kinase complex with Pho85p which phosphorylates Mmr1p and is regulated by Pho81p; involved in glycogen metabolism, expression is cell-cycle regulated; Pcl7p [Saccharomyces cerevisiae] 129 322 8 PHE0004432_PMON94112 0 100 15156338 ref|NP_354295.1| hypothetical protein AGR_C_2368 [Agrobacterium tumefaciens str. C58] 130 323 8 PHE0004472_PMON94115 1.00E−128 100 16323494 ref|NP_187978.1|seven in absentia (SINA) family protein [Arabidopsis thaliana] 131 324 14 PHE0004472_PMON94126 1.00E−128 100 16323494 ref|NP_187978.1|seven in absentia (SINA) family protein [Arabidopsis thaliana] 132 325 14 PHE0004488_PMON95609 1.00E−123 100 21554344 ref|NP_198627.1|ASF1- like anti-silencing family protein [Arabidopsis thaliana] 133 326 14 PHE0004491_PMON95628 3.00E−12 45 14916641 dbj|BAB19648.1| preprophytosulfokine [Oryza sativa] 134 327 14 PHE0004492_PMON95614 0 100 22331730 ref|NP_190653.2|phototropic- responsive NPH3 family protein [Arabidopsis thaliana] 135 328 10 PHE0004545_PMON95117 1.00E−106 100 28973235 ref|NP_173200.1| ribosomal protein L14 family protein [Arabidopsis thaliana] 136 329 8 PHE0004574_PMON94433 0 100 16329404 ref|NP_440132.1|transaldolase [Synechocystis sp. PCC 6803] 137 330 14 PHE0004606_PMON95627 0 100 130709 pir||S29317 phosphoprotein phosphatase (EC 3.1.3.16) 1 - maize gb|AAA33545.1| protein phosphatase-1 138 331 8 PHE0004620_PMON94189 1.00E−101 57 56421275 ref|YP_148593.1|6- phosphofructokinase (phosphofructokinase) (phosphohexokinase) [Geobacillus kaustophilus HTA426] 139 332 14 PHE0004620_PMON94442 1.00E−101 57 56421275 ref|YP_148593.1|6- phosphofructokinase (phosphofructokinase) (phosphohexokinase) [Geobacillus kaustophilus HTA426] 140 333 14 PHE0004622_PMON95621 0 100 10177836 ref|NP_974942.1|F-box family protein [Arabidopsis thaliana] 141 334 8 PHE0004626_PMON95101 0 88 50942161 ref|XP_480608.1|putative gamma-aminobutyrate transaminase subunit precursor isozyme 3 [Oryza sativa (japonica cultivar- group)] 142 335 8 PHE0004630_PMON94367 0 100 7270516 emb|CAB80281.1|NAD+ dependent isocitrate dehydrogenase-like protein [Arabidopsis thaliana] 143 336 3 PHE0004634_PMON94385 1.00E−102 100 61656127 ref|NP_176491.1|AP2 domain-containing transcription factor, putative [Arabidopsis thaliana] 144 337 2 PHE0004640_PMON95066 0 73 34913436 ref|NP_918065.1|putative fatty acid condensing enzyme CUT1 [Oryza sativa (japonica cultivar- group)] 145 338 8 PHE0004645_PMON94655 1.00E−136 100 18411867 ref|NP_565174.1|14-3-3 protein GF14 pi (GRF13) [Arabidopsis thaliana] 146 339 14 PHE0004645_PMON94685 1.00E−136 100 18411867 ref|NP_565174.1|14-3-3 protein GF14 pi (GRF13) [Arabidopsis thaliana] 147 340 8 PHE0004647_PMON94651 1.00E−117 100 21554066 pir||T02447 hypothetical protein At2g46000 Arabidopsis thaliana 148 341 14 PHE0004647_PMON94688 1.00E−117 100 21554066 gb|AAM63147.1|unknown [Arabidopsis thaliana] 149 342 14 PHE0004650_PMON94686 1.00E−112 100 67633514 gb|AAY78681.1|putative E3 ubiquitin ligase SCF complex subunit SKP1/ASK1 [Arabidopsis thaliana] 150 343 8 PHE0004652_PMON94657 1.00E−138 100 38603872 dbj|BAD43212.1|putative glutamate/aspartate-binding peptide [Arabidopsis thaliana] 151 344 14 PHE0004652_PMON94687 1.00E−138 100 38603872 dbj|BAD43212.1|putative glutamate/aspartate-binding peptide [Arabidopsis thaliana] 152 345 8 PHE0004687_PMON94669 7.00E−61 91 21592528 ref|NP_568396.1|ring-box protein-related [Arabidopsis thaliana] 153 346 10 PHE0004689_PMON95131 0 100 7268004 emb|CAB78344.1|serine/threonine- specific protein kinase MHK [Arabidopsis thaliana] 154 347 10 PHE0004691_PMON95129 0 100 51978966 emb|CAB61629.1| spermidine synthase 1 [Oryza sativa] 155 348 14 PHE0004719_PMON94698 1.00E−147 100 28416631 ref|NP_564556.1|zinc finger (C3HC4-type RING finger) family protein [Arabidopsis thaliana] 156 349 8 PHE0004719_PMON95089 1.00E−147 100 28416631 ref|NP_564556.1|zinc finger (C3HC4-type RING finger) family protein [Arabidopsis thaliana] 157 350 8 PHE0004734_PMON94667 1.00E−87 100 5080771 ref|NP_172848.1| eukaryotic translation initiation factor 5A-1/eIF- 5A 1 [Arabidopsis thaliana] 158 351 10 PHE0004735_PMON95116 9.00E−88 100 21592652 ref|NP_177100.1| eukaryotic translation initiation factor 5A, putative/ eIF-5A, putative [Arabidopsis thaliana] 159 352 8 PHE0004739_PMON95110 1.00E−109 100 6562282 emb|CAB62652.1|rac-like GTP binding protein Arac11 [Arabidopsis thaliana] 160 353 8 PHE0004753_PMON95105 0 100 6684442 ref|NP_178062.1| succinate-semialdehyde dehydrogenase (SSADH1) [Arabidopsis thaliana] 161 354 8 PHE0004759_PMON95109 0 100 29824301 ref|NP_849582.1|expressed protein [Arabidopsis thaliana] 162 355 10 PHE0004770_PMON95122 1.00E−32 92 51038072 gb|AAT93875.1|unknown protein [Oryza sativa (japonica cultivar-group)] 163 356 10 PHE0004772_PMON95132 6.00E−36 33 9758946 ref|NP_200265.1| expressed protein [Arabidopsis thaliana] 164 357 10 PHE0004774_PMON95147 6.00E−52 66 50909195 ref|XP_466086.1|putative multiple stress-responsive zinc-finger protein [Oryza sativa (japonica cultivar- group)] 165 358 10 PHE0004777_PMON95118 2.00E−64 100 26452894 ref|NP_180514.1|DNA- directed RNA polymerase I(A) and III(C) 14 kDa subunit (RPAC14) [Arabidopsis thaliana] 166 359 14 PHE0004785_PMON95057 1.00E−145 84 34484312 sp|Q6UNT2|RL5_CUCSA 60S ribosomal protein L5 167 360 10 PHE0004786_PMON95604 0 100 7267537 ref|NP_192634.1| phosphate-responsive protein, putative (EXO) [Arabidopsis thaliana] 168 361 8 PHE0004788_PMON95092 0 84 31126776 ref|XP_506910.1| PREDICTED OSJNBa0057G07.4 gene product [Oryza sativa (japonica cultivar-group)] 169 362 10 PHE0004799_PMON95602 0 99 9843858 emb|CAC03739.1|flavin containing polyamine oxidase [Zea mays] 170 363 10 PHE0004841_PMON95636 0 100 50909767 ref|XP_466372.1|cryptochrome 1a [Oryza sativa (japonica cultivar-group)] 171 364 10 PHE0004844_PMON95637 3.00E−53 100 62734659 gb|AAX96768.1|expressed protein [Oryza sativa (japonica cultivar-group)] 172 365 14 PHE0004854_PMON95611 1.00E−163 100 21592743 ref|NP_199265.1|ribose 5- phosphate isomerase-related [Arabidopsis thaliana] 173 366 10 PHE0004862_PMON95601 5.00E−56 100 34902924 dbj|BAB07982.1|FPF1 protein-like [Oryza sativa (japonica cultivar-group)] 174 367 10 PHE0004888_PMON95603 0 100 32405610 ref|XP_323418.1|hypothetical protein [Neurospora crassa] 175 368 n/a At1g21790.1 1.00E−168 100 21593249 ref|NP_564152.1|expressed protein [Arabidopsis thaliana] 176 369 n/a ERD4 0 100 17104683 ref|NP_564354.1|early- responsive to dehydration stress protein (ERD4) [Arabidopsis thaliana] 177 370 n/a At1g78070.2 0 100 42572153 ref|NP_974167.1|WD-40 repeat family protein [Arabidopsis thaliana] 178 371 n/a At1g78070.1 1.00E−128 100 18411805 ref|NP_565168.1|WD-40 repeat family protein [Arabidopsis thaliana] 179 372 n/a At3g47340.1 0 100 5541701 ref|NP_190318.1| asparagine synthetase 1 [glutamine-hydrolyzing]/ glutamine-dependent asparagine synthetase 1 (ASN1) [Arabidopsis thaliana] 180 373 n/a At3g47340.3 0 100 30692853 ref|NP_850664.1|asparagine synthetase 1 [glutamine- hydrolyzing]/glutamine- dependent asparagine synthetase 1 (ASN1) [Arabidopsis thaliana] 181 374 n/a At3g47340.2 0 100 30692849 ref|NP_850663.1|asparagine synthetase 1 [glutamine- hydrolyzing]/glutamine- dependent asparagine synthetase 1 (ASN1) [Arabidopsis thaliana] 182 375 n/a At5g13170.1 1.00E−163 100 9955561 ref|NP_196821.1|nodulin MtN3 family protein [Arabidopsis thaliana] 183 376 n/a At2g19900.1 0 100 28059162 ref|NP_179580.1|malate oxidoreductase, putative [Arabidopsis thaliana] 184 377 n/a At5g09480.1 8.00E−80 100 9955535 ref|NP_196510.1| hydroxyproline-rich glycoprotein family protein [Arabidopsis thaliana] 185 378 n/a At5g09530.1 0 100 7671436 ref|NP_196515.1| hydroxyproline-rich glycoprotein family protein [Arabidopsis thaliana] 186 379 n/a At2g42790.1 0 100 21700853 ref|NP_181807.1|citrate synthase, glyoxysomal, putative [Arabidopsis thaliana] 187 380 n/a At3g56200.1 0 100 7572918 ref|NP_191179.1|amino acid transporter family protein [Arabidopsis thaliana] 188 381 n/a At5g01520.1 1.00E−141 100 7327811 ref|NP_195772.1|zinc finger (C3HC4-type RING finger) family protein [Arabidopsis thaliana] 189 382 n/a At5g01520.2 2.00E−97 100 7327811 ref|NP_195772.1|zinc finger (C3HC4-type RING finger) family protein [Arabidopsis thaliana] 190 383 n/a At5g66780.1 2.00E−66 100 9758128 d ref|NP_201479.1| expressed protein [Arabidopsis thaliana] 191 384 n/a At5g59320.1 1.00E−61 100 24417292 ref|NP_568905.1|lipid transfer protein 3 (LTP3) [Arabidopsis thaliana] 192 385 n/a AtHB7 1.00E−151 100 20259175 gb|AAM14303.1|putative homeodomain transcription factor protein ATHB-7 [Arabidopsis thaliana] 193 386 n/a RD20 1.00E−136 100 20465881 ref|NP_180896.1|calcium- binding RD20 protein (RD20) [Arabidopsis thaliana] Table 1 provides a list of protein encoding DNA (“genes”) that are useful as recombinant DNA for production of transgenic plants with enhanced agronomic trait, the elements of Table 1 are described by reference to: “NUC SEQ ID NO” which is a SEQ ID NO for a DNA sequence in the Sequence Listing. “PEP SEQ ID NO” which is a SEQ ID NO for an amino acid sequence in the Sequence Listing. GENE ID” which is an arbitrary name for the recombinant DNA. “Base Vector” which is a reference to the identifying number in Table 5 of base vectors used for transformation of the recombinant DNA. Construction of plant transformation constructs is illustrated in Example 1. “annotation” refers to a description of the top hit protein obtained from an amino acid sequence query of each PEP SEQ ID NO to GenBank database of the National Center for Biotechnology Information (NCBI). Identifier is the GenBank ID number for the informative BLAST hit with -FT.

Screening Methods for Transgenic Plants with Enhanced Agronomic Trait

Many transgenic events which survive to fertile transgenic plants that produce seeds and progeny plants will not exhibit an enhanced agronomic trait. Screening is necessary to identify the transgenic plant of this invention. Transgenic plants having enhanced agronomic traits are identified from populations of plants transformed as described herein by evaluating the trait in a variety of assays to detect an enhanced agronomic trait. These assays also may take many forms, including but not limited to, analyses to detect changes in the chemical composition, biomass, physiological properties, morphology of the plant. Changes in chemical compositions such as nutritional composition of grain can be detected by analysis of the seed composition and content of protein, free amino acids, oil, free fatty acids, starch or tocopherols. Changes in biomass characteristics can be made on greenhouse or field grown plants and can include plant height, stem diameter, root and shoot dry weights; and, for corn plants, ear length and diameter. Changes in physiological properties can be identified by evaluating responses to stress conditions, e.g., assays using imposed stress conditions such as water deficit, nitrogen deficiency, cold growing conditions, pathogen or insect attack or light deficiency, or increased plant density. Changes in morphology can be measured by visual observation of tendency of a transformed plant with an enhanced agronomic trait to also appear to be a normal plant as compared to changes toward bushy, taller, thicker, narrower leaves, striped leaves, knotted trait, chlorosis, albino, anthocyanin production, or altered tassels, ears or roots. Other screening properties include days to pollen shed, days to silking, leaf extension rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace roots, stay green, stalk lodging, root lodging, plant health, barreness/prolificacy, green snap, and pest resistance. In addition, phenotypic characteristics of harvested grain may be evaluated, including number of kernels per row on the ear, number of rows of kernels on the ear, kernel abortion, kernel weight, kernel size, kernel density and physical grain quality.

Although preferred seeds for transgenic plants with enhanced agronomic traits of this invention are corn and soybean plants, other seeds are for cotton, canola, wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass

EXAMPLE Example 1 Plant Expression Constructs

This example illustrates the construction of plasmids for transferring recombinant DNA into plant cells which can be regenerated into transgenic plants of this invention.

Primers for PCR amplification of protein coding nucleotides of recombinant DNA are designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions. Each recombinant DNA coding for a protein identified in Table 1 is amplified by PCR prior to insertion into the insertion site of one of the base vectors as referenced in Table 5.

A. Corn Transformation Constructs

With reference to Table 2 and FIG. 1, pMON82060 illustrates the elements of base vector 1 for corn transformation. Other base vectors for corn transformation were also constructed by replacing the gene of interest plant expression cassette elements of base vector 1, i.e. the promoter, leader, intron and terminator elements, with the elements listed in Table 5 to provide base vectors 2-12 for corn transformation. Each of the protein encoding DNA as identified in Table 1 is placed in the gene of interest plant expression cassette before the termination sequence in each of the base vector 1-12.

TABLE 2 pMON82060 Coordinates of SEQ ID function name annotation NO: 12603 Agro B-AGRtu.right border Agro right border sequence, essential for 5235-5591 transformation transfer of T-DNA. Gene of P-Os.Act1 Promoter from the rice actin gene act1. 5609-7009 interest plant L-Os.Act1 Leader (first exon) from the rice actin 1 expression gene. cassette I-Os.Act1 First intron and flanking UTR exon sequences from the rice actin 1 gene T-St.Pis4 The 3′ non-translated region of the 7084-8026 potato proteinase inhibitor II gene which functions to direct polyadenylation of the mRNA Plant P-CaMV.35S CaMV 35S promoter 8075-8398 selectable L-CaMV.35S 5′ UTR from the 35S RNA of CaMV marker CR-Ec.nptII-Tn5 nptII selectable marker that confers 8432-9226 expression resistance to neomycin and kanamycin cassette T-AGRtu.nos A 3′ non-translated region of the 9255-9507 nopaline synthase gene of Agrobacterium tumefaciens Ti plasmid which functions to direct polyadenylation of the mRNA . . . Agro B-AGRtu.left border Agro left border sequence, essential for  39-480 transformation transfer of T-DNA. Maintenance OR-Ec.oriV-RK2 The vegetative origin of replication from 567-963 in E. coli plasmid RK2. CR-Ec.rop Coding region for repressor of primer 2472-2663 from the ColE1 plasmid. Expression of this gene product interferes with primer binding at the origin of replication, keeping plasmid copy number low. OR-Ec.ori-ColE1 The minimal origin of replication from 3091-3679 the E. coli plasmid ColE1. P-Ec.aadA-SPC/STR promoter for Tn7 adenylyltransferase 4210-4251 (AAD(3″)) CR-Ec.aadA- Coding region for Tn7 4252-5040 SPC/STR adenylyltransferase (AAD(3″)) conferring spectinomycin and streptomycin resistance. T-Ec.aadA-SPC/STR 3′ UTR from the Tn7 adenylyltransferase 5041-5098 (AAD(3″)) gene of E. coli.

Elements of a corn transformation plasmid, pMON17730, for expressing a Leuconostoc mesenteroides sucrose phosphorylase are illustrated in Table 3. This construct was assembled using the technology known in the art.

TABLE 3 pMON17730 Coordinates of function name annotation SEQ ID NO: 12606 Agro B-AGRtu.right Agro right border sequence, essential 4862-5218 transformation border for transfer of T-DNA. Gene of P-Zm.Brittle2 Promoter from thecorn brittle 2 gene interest plant L-Zm.Brittle2 5′ untranslated region from the corn expression brittel 2 gene. cassette L-Ta.Lhcb1 wheat CAB leader I-Os.Act1 First intron and flanking UTR exon 5276-6375 sequences from the rice actin 1 gene CR-Lm.sp11 PHE0004028_PMON17730 SPL 6385-7857 coding region T-Ta.Hsp17 The 3′ non-translated region of the 7870-8079 wheat low molecular weight heat shock protein gene Plant P-CaMV.35S CaMV 35S promoter 8226-8518 selectable CR-Ec.nptII- nptII selectable marker that confers 8583-9377 marker Tn5 resistance to neomycin and expression kanamycin cassette T-AGRtu.nos A 3′ non-translated region of the 9409-9661 nopaline synthase gene of Agrobacterium tumefaciens Ti plasmid which functions to direct polyadenylation of the mRNA . . . Agro B-AGRtu.left Agro left border sequence, essential 10003-10026 transformation border for transfer of T-DNA. Maintenance OR-Ec.oriV- The vegetative origin of replication 194-590 in E. coli RK2 from plasmid RK2. CR-Ec.rop Coding region for repressor of 2099-2290 primer from the ColE1 plasmid. Expression of this gene product interferes with primer binding at the origin of replication, keeping plasmid copy number low. OR-Ec.ori- The minimal origin of replication 2718-3306 ColE1 from the E. coli plasmid ColE1. P-Ec.aadA- promoter for Tn7 3837-3878 SPC/STR adenylyltransferase (AAD(3″)) CR-Ec.aadA- Coding region for Tn7 3879-4667 SPC/STR adenylyltransferase (AAD(3″)) conferring spectinomycin and streptomycin resistance. T-Ec.aadA- 3′ UTR from the Tn7 4668-4725 SPC/STR adenylyltransferase (AAD(3″)) gene of E. coli.

B. Soybean Transformation Constructs

Plasmids for use in transformation of soybean are also prepared. Elements of an exemplary common expression vector plasmid pMON82053 are shown in Table 4 and FIG. 2. Other base vectors for soybean transformation were also constructed by replacing the gene of interest plant expression cassette elements of base vector 13, i.e. the promoter, leader, intron and terminator elements, with the elements listed in Table 5 to provide base vectors 13-15 for soybean transformation. Each of the protein encoding DNA as identified in Table 1 is placed in the gene of interest plant expression cassette before the termination sequence in each of the base vector 13-15.

TABLE 4 pMON82053 Coordinates of SEQ ID function name annotation NO: 12604 Agro B-AGRtu.left border Agro left border 6144-6585 transforamtion sequence, essential for transfer of T-DNA. Plant P-At.Act7 Promoter from the 6624-7861 selectable arabidopsis actin 7 gene marker L-At.Act7 5′UTR of Arabidopsis expression Act7 gene cassette I-At.Act7 Intron from the Arabidopsis actin 7 gene TS-At.ShkG-CTP2 Transit peptide region of 7864-8091 Arabidopsis EPSPS CR-AGRtu.aroA- Synthetic CP4 coding 8092-9459 CP4.nno_At region with dicot preferred codon usage. T-AGRtu.nos A 3′ non-translated region 9466-9718 of the nopaline synthase gene of Agrobacterium tumefaciens Ti plasmid which functions to direct polyadenylation of the mRNA. Gene of P-CaMV.35S-enh Promoter for 35S RNA  1-613 interest from CaMV containing a expression duplication of the −90 to −350 cassette region. T-Gb.E6-3b 3′ untranslated region  688-1002 from the fiber protein E6 gene of sea-island cotton; Agro B-AGRtu.right border Agro right border 1033-1389 transformation sequence, essential for transfer of T-DNA. Maintenance OR-Ec.oriV-RK2 The vegetative origin of 5661-6057 in E. coli replication from plasmid RK2. CR-Ec.rop Coding region for 3961-4152 repressor of primer from the ColE1 plasmid. Expression of this gene product interferes with primer binding at the origin of replication, keeping plasmid copy number low. OR-Ec.ori-ColE1 The minimal origin of 2945-3533 replication from the E. coli plasmid ColE1. P-Ec.aadA-SPC/STR romoter for Tn7 2373-2414 adenylyltransferase (AAD(3″)) CR-Ec.aadA- Coding region for Tn7 1584-2372 SPC/STR adenylyltransferase (AAD(3″)) conferring spectinomycin and streptomycin resistance. T-Ec.aadA-SPC/STR 3′ UTR from the Tn7 1526-1583 adenylyltransferase (AAD(3″)) gene of E. coli.

TABLE 5 Compositions of expression cassettes for gene of interest in plant transformation base vectors SEQ SEQ SEQ SEQ ID ID ID ID promoter NO leader NO intron NO terminator NO Base vector for corn 1 P-Os.Act1 12581 L-Os.Act1 12592 I-Os.Act1 12596 T-St.Pis4 12598 2 P-Hv.Per1 12582 L-Hv.Per1 12593 I-Zm.DnaK 12597 T-St.Pis4 12598 3 P-Zm.RAB17 12591 NONE / I-Zm.DnaK 12597 T-St.Pis4 12598 4 P-Zm.NAS2 12584 L-Zm.NAS2 12595 I-Zm.DnaK 12597 T-St.Pis4 12598 5 P-Zm.PPDK 12585 L-Zm.PPDK 12588 I-Zm.DnaK 12597 T-St.Pis4 12598 6 P-Os.GT1 12586 NONE / I-Zm.DnaK 12597 T-St.Pis4 12598 7 P-Zm.PPDK 12587 L-Zm.PPDK 12588 I-Zm.DnaK 12597 T-St.Pis4 12600 8 P-Os.Act1 12581 L-Os.Act1 12592 I-Os.Act1 12597 T-St.Pis4 12598 9 P-Zm.PPDK 12587 L-Zm.PPDK 12588 I-Zm.DnaK 12597 T-St.Pis4 12600 10  P-Os.Act1 12581 L-Os.Act1 12592 I-Os.Act1 12596 T-St.Pis4 12598 11  P-Zm.SzeinC1 12589 L- 12601 I-Zm.DnaK 12597 T-St.Pis4 12598 Zm.SzeinC1 12  P-Zm.NAS2 12584 L-Zm.NAS2 12595 I-Zm.DnaK 12597 T-St.Pis4 12598 Base vector for Soybean 13  P-CaMV.35S- 12590 NONE / NONE / T-Gb.E6 12599 enh 14  P-CaMV.35S- 12590 NONE / NONE / T-Gb.E6 12599 enh 15  P-Gm.Sphas 1 12583 L- 12594 NONE / T-Gb.E6 12599 Gm.Sphas1 DNA constructs with some recombinant DNA of interest, e.g., SEQ ID NO: 72, also contain a chloroplast transit peptide adjacent to the recombinant DNA.

C. Cotton Transformation Vector

Plasmids for use in transformation of cotton are also prepared. Elements of an exemplary common expression vector plasmid pMON99053 are shown in Table 6 below and FIG. 3. Primers for PCR amplification of protein coding nucleotides of recombinant DNA are designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions. Each recombinant DNA coding for a protein identified in Table 1 is amplified by PCR prior to insertion into the insertion site within the gene of interest expression cassette of pMON99053

TABLE 6 Coordinates of SEQ ID NO: function name annotation 12606 Agro B-AGRtu.right border Agro right border sequence, 11364-11720 transforamtion essential for transfer of T-DNA. Gene of interest Exp-CaMV.35S- Enhanced version of the 35S RNA 7794-8497 expression enh + ph.DnaK promoter from CaMV plus the cassette petunia hsp70 5′ untranslated region T-Ps.RbcS2-E9 The 3′ non-translated region of the  67-699 pea RbcS2 gene which functions to direct polyadenylation of the mRNA. Plant selectable Exp-CaMV.35S Promoter from the rice actin 1 gene  730-1053 marker CR-Ec.nptII-Tn5 first exon of the rice actin 1 gene 1087-1881 expression T-AGRtu.nos A 3′ non-translated region of the 1913-2165 cassette nopaline synthase gene of Agrobacterium tumefaciens Ti plasmid which functions to direct polyadenylation of the mRNA. Agro B-AGRtu.left border Agro left border sequence, essential 2211-2652 transformation for transfer of T-DNA. Maintenance in OR-Ec.oriV-RK2 The vegetative origin of replication 2739-3135 E. coli from plasmid RK2. CR-Ec.rop Coding region for repressor of primer 4644-4835 from the ColE1 plasmid. Expression of this gene product interferes with primer binding at the origin of replication, keeping plasmid copy number low. OR-Ec.ori-ColE1 The minimal origin of replication 5263-5851 from the E. coli plasmid ColE1. P-Ec.aadA-SPC/STR romoter for Tn7 adenylyltransferase 6382-6423 (AAD(3″)) CR-Ec.aadA-SPC/STR Coding region for Tn7 6424-7212 adenylyltransferase (AAD(3″)) conferring spectinomycin and streptomycin resistance. T-Ec.aadA-SPC/STR 3′ UTR from the Tn7 7213-7270 adenylyltransferase (AAD(3″)) gene of E. coli.

Example 2 Corn Plant Transformation

This example illustrates the production and identification of transgenic corn cells in seed of transgenic corn plants having an enhanced agronomic trait, i.e. enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, enhanced tolerance to cold and/or improved seed compositions as compared to control plants. Transgenic corn cells are prepared with recombinant DNA expressing each of the protein encoding DNAs listed in Table 1 by Agrobacterium-mediated transformation using the corn transformation vectors 1-12 prepared as disclosed in Example 1. Corn transformation is effected using methods disclosed in U.S. Patent Application Publication 2004/0344075 A1 where corn embryos are inoculated and co-cultured with the Agrobacterium tumefaciens strain ABI and the corn transformation vector. To regenerate transgenic corn plants the transgenic callus resulting from transformation is placed on media to initiate shoot development in plantlets which are transferred to potting soil for initial growth in a growth chamber followed by a mist bench before transplanting to pots where plants are grown to maturity. The plants are self fertilized and seed is harvested for screening as seed, seedlings or progeny R2 plants or hybrids, e.g., for yield trials in the screens indicated above.

Many transgenic events which survive to fertile transgenic plants that produce seeds and progeny plants do not exhibit an enhanced agronomic trait. The transgenic plants and seeds having the transgenic cells of this invention which have recombinant DNA imparting the enhanced agronomic traits are identified by screening for nitrogen use efficiency, yield, water use efficiency, cold tolerance and improved seed composition.

Example 3 Soybean Plant Transformation

This example illustrates the production and identification of transgenic soybean cells in seed of transgenic soybean plants having an enhanced agronomic trait, i.e. enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, enhanced tolerance to cold and/or improved seed compositions as compared to control plants. Transgenic soybean cells are prepared with recombinant DNA expressing each of the protein encoding DNAs listed in Table 1 by Agrobacterium-mediated transformation using the soybean transformation vectors 13-15 prepared as disclosed in Example 1. Soybean transformation is effected using methods disclosed in U.S. Pat. No. 6,384,301 where soybean meristem explants are wounded then inoculated and co-cultured with the soybean transformation vector, then transferred to selection media for 6-8 weeks to allow selection and growth of transgenic shoots.

The transformation is repeated for each of the protein encoding DNAs identified in Table 1 in one of the base vectors 13-15.

Transgenic shoots producing roots are transferred to the greenhouse and potted in soil. Many transgenic events which survive to fertile transgenic plants that produce seeds and progeny plants do not exhibit an enhanced agronomic trait. The transgenic plants and seeds having the transgenic cells of this invention which have recombinant DNA imparting the enhanced agronomic traits are identified by screening for nitrogen use efficiency, yield, water use efficiency, cold tolerance and improved seed composition.

Example 4 Cotton Transgenic Plants with Enhanced Agronomic Traits

Cotton transformation is performed as generally described in WO0036911 and in U.S. Pat. No. 5,846,797. Transgenic cotton plants containing the recombinant DNA having a sequence of SEQ ID NO: 1 through SEQ ID NO: 193 are obtained by transforming with the cotton transformation vector identified in Example 1.

Progeny transgenic plants are selected from a population of transgenic cotton events under specified growing conditions and are compared with control cotton plants. Control cotton plants are substantially the same cotton genotype but without the recombinant DNA, for example, either a parental cotton plant of the same genotype that was not transformed with the identical recombinant DNA or a negative isoline of the transformed plant. Additionally, a commercial cotton cultivar adapted to the geographical region and cultivation conditions, i.e. cotton variety ST474, cotton variety FM 958, and cotton variety Siokra L-23, are used to compare the relative performance of the transgenic cotton plants containing the recombinant DNA. The specified culture conditions are growing a first set of transgenic and control plants under “wet” conditions, i.e. irrigated in the range of 85 to 100 percent of evapotranspiration to provide leaf water potential of −14 to −18 bars, and growing a second set of transgenic and control plants under “dry” conditions, i.e. irrigated in the range of 40 to 60 percent of evapotranspiration to provide a leaf water potential of −21 to −25 bars. Pest control, such as weed and insect control is applied equally to both wet and dry treatments as needed. Data gathered during the trial includes weather records throughout the growing season including detailed records of rainfall; soil characterization information; any herbicide or insecticide applications; any gross agronomic differences observed such as leaf morphology, branching habit, leaf color, time to flowering, and fruiting pattern; plant height at various points during the trial; stand density; node and fruit number including node above white flower and node above crack boll measurements; and visual wilt scoring. Cotton boll samples are taken and analyzed for lint fraction and fiber quality. The cotton is harvested at the normal harvest timeframe for the trial area. Enhanced water use efficiency is indicated by increased yield, improved relative water content, enhanced leaf water potential, increased biomass, enhanced leaf extension rates, and improved fiber parameters.

Cotton plants with the transgenic cells by this invention are identified from among the transgenic cotton plants by agronomic trait screening as having increased yield and enhanced water use efficiency.

Example 5 Homolog Identification

This example illustrates the identification of homologs of proteins encoded by the DNA identified in Table 1 which is used to provide transgenic seed and plants having enhanced agronomic traits. From the sequence of the homologs, homologous DNA sequence can be identified for preparing additional transgenic seeds and plants of this invention with enhanced agronomic traits.

An “All Protein Database” was constructed of known protein sequences using a proprietary sequence database and the National Center for Biotechnology Information (NCBI) non-redundant amino acid database (nr.aa). For each organism from which a polynucleotide sequence provided herein was obtained, an “Organism Protein Database” was constructed of known protein sequences of the organism; it is a subset of the All Protein Database based on the NCBI taxonomy ID for the organism.

The All Protein Database was queried using amino acid sequences provided herein as SEQ ID NO: 194 through SEQ ID NO: 386 using NCBI “blastp” program with E-value cutoff of 1e-8. Up to 1000 top hits were kept, and separated by organism names. For each organism other than that of the query sequence, a list was kept for hits from the query organism itself with a more significant E-value than the best hit of the organism. The list contains likely duplicated genes of the polynucleotides provided herein, and is referred to as the Core List. Another list was kept for all the hits from each organism, sorted by E-value, and referred to as the Hit List.

The Organism Protein Database was queried using polypeptide sequences provided herein as SEQ ID NO: 194 through SEQ ID NO: 386 using NCBI “blastp” program with E-value cutoff of 1e-4. Up to 1000 top hits were kept. A BLAST searchable database was constructed based on these hits, and is referred to as “SubDB”. SubDB was queried with each sequence in the Hit List using NCBI “blastp” program with E-value cutoff of 1e-8. The hit with the best E-value was compared with the Core List from the corresponding organism. The hit is deemed a likely ortholog if it belongs to the Core List, otherwise it is deemed not a likely ortholog and there is no further search of sequences in the Hit List for the same organism. Homologs from a large number of distinct organisms were identified and are reported by amino acid sequences of SEQ ID NO: 387 through SEQ ID NO: 12580. These relationships of proteins of SEQ ID NO: 194 through 386 and homologs of SEQ ID NO: 387 through 12580 is identified in Table 7. The source organism for each homolog is found in the Sequence Listing.

TABLE 7 SEQ ID NO: homolog SEQ ID NOs 196: 3549 1976 8970 12287 11799 758 6083 9821 8256 7610 7869 4091 1111 1113 8630 7054 10917 3094 6712 9080 2702 2718 1130 1131 5382 6582 559 2169 1134 1132 1139 2295 11615 8090 2133 5063 5000 10336 12279 3828 7214 1485 2156 2232 2229 2242 2209 2203 2177 2207 2160 2151 11166 3220 197: 3549 1976 4850 8970 12287 11799 758 6083 9821 4946 11935 8256 7610 7869 1841 9456 4091 1113 1111 8630 7054 7880 6876 6237 6712 9080 2702 2718 1130 1131 5382 6582 559 2169 1134 1132 1139 2295 11615 8090 5063 5000 10336 12279 3828 7214 1485 2156 2232 2229 2242 2209 2203 2207 2177 2160 2151 7622 1377 6970 6143 198: 3549 1976 2210 6154 1028 1769 758 12325 9821 2973 4946 11935 8256 7610 5387 5384 5361 10434 8983 5051 4091 2766 6248 1113 1111 8630 9080 2702 2718 1131 1130 5382 7052 6582 1134 1132 1139 11615 2295 8090 6572 4803 1970 8113 3883 9565 1707 517 12372 11514 5441 5421 3828 7214 1485 1097 199: 3549 4850 2210 8970 12287 2360 11500 11799 6912 1028 6154 758 5783 6083 9552 12325 9821 2973 4946 8256 7610 5387 5361 5384 5300 10434 8983 5051 1111 1113 8630 9080 2702 2718 1130 1131 5382 7052 6582 559 2169 1134 1132 1139 8090 6572 11350 7138 1730 10762 11345 527 8679 5063 5000 2879 517 7986 12372 11514 10336 6955 12279 5441 5421 3828 7214 1485 2229 2207 2242 2232 2209 2203 2156 2160 2151 5328 8248 200: 11500 5617 8150 3321 2181 4364 1769 1028 5122 11328 6042 2711 1760 4874 4098 1914 11853 7334 6504 10624 2638 11705 7913 12171 12198 10430 12189 12219 10404 10432 10408 6957 8282 6184 11935 580 10470 1940 11039 8629 1096 742 12505 5801 11671 4006 12473 6778 2607 10849 6279 7500 2657 5584 8059 2622 2043 3269 10363 6186 9631 9243 11098 1168 6690 8584 10577 687 2977 9804 9337 6306 9118 4356 10225 9740 6652 5251 12514 7463 706 3048 3780 1925 11765 9803 10824 3004 5275 8642 1664 12173 4049 2031 11681 8980 2339 9172 11955 10576 9333 10482 813 5656 4628 10843 2352 5484 2856 4313 2877 1633 11143 6066 7722 7746 10941 11741 2941 2745 11364 7638 7884 1328 5606 6580 11262 7483 8156 412 453 7288 6842 1286 7896 9734 6570 10595 8863 1246 7112 12464 1373 3779 2705 5044 4017 5712 4619 3539 1029 1610 5976 4964 11724 9037 8989 1126 4073 395 10344 5428 4845 1611 10484 4496 3517 3418 10294 2427 3442 9747 5534 9571 1125 9720 9319 12346 3417 1588 2779 4611 5312 10179 6867 3049 3051 9900 1265 9463 4576 764 6024 432 8921 11379 2141 1755 9498 7395 8179 7462 7279 8729 9676 11351 1758 10907 4995 1205 608 12100 8331 8341 10326 6852 11947 6597 2475 6407 8077 10788 11815 5269 489 9317 5574 11240 11821 11485 2868 9753 676 11223 1924 8045 1689 12035 11980 5906 7805 6728 5177 1711 1715 5050 1601 11242 1010 11286 7814 7152 3730 5888 615 11078 9681 2883 8522 8210 4450 11632 7573 6031 2713 3861 9480 5307 7874 2048 5136 8625 2168 4580 10634 5772 5082 8731 2678 9311 10561 7803 4408 6227 12026 11234 7247 5578 9683 3999 2953 2193 3370 11542 10711 6403 4207 11251 8447 6805 727 951 737 9090 1828 1928 2277 986 739 7044 10025 7409 9449 944 8427 10911 3965 1299 5294 6332 5145 9418 6150 9008 1004 3831 5157 6968 11922 7392 9855 5061 5448 6857 2354 2879 620 7986 10208 4520 9003 8015 525 8013 11884 10726 12493 9260 8508 5693 1450 4258 201: 7470 10842 5790 6772 1530 9966 9973 10368 655 4677 4157 1015 9967 9732 1621 1702 2553 11599 9342 3724 6613 4462 2681 4577 3827 8039 2557 8538 9605 12321 3228 2139 9255 11428 3022 5404 9564 12166 8047 11255 11888 1492 5870 4250 5541 2481 8585 5674 2062 2021 6718 2810 4015 12306 8941 3135 7850 7009 4247 5760 643 2512 2422 8709 5661 2437 11487 8706 3703 6811 5006 6000 2290 11973 8426 2912 6498 10642 8257 5362 1189 996 1740 10904 5778 4372 12095 1616 9708 1598 4525 7513 1934 10939 9044 7273 6105 6950 12122 5936 2802 3711 8640 6644 9842 6994 2587 7510 8609 1877 5408 8009 9943 8475 4333 8476 2651 5379 11144 202: 7477 1676 4448 2400 6045 6940 8526 9923 11995 8913 10513 634 7969 11746 6446 4371 1018 4026 10874 11604 5505 9219 4140 11205 12025 3605 1669 1987 2822 2279 10124 11930 4546 3504 1950 7696 1604 4492 710 11737 3171 8574 11646 9030 765 203: 9581 1789 9205 10127 507 7859 5085 10794 2201 5072 1384 7541 12225 5253 4000 8561 1469 3834 12504 9837 7137 4670 9143 1972 230 11807 7457 3867 12503 9644 10286 686 3416 8708 913 9391 9343 1949 971 11938 12315 7511 9076 8346 3455 1790 6685 11054 10989 4775 9544 2197 3225 1198 7996 9715 6751 11217 3189 10361 3589 2768 4753 393 7426 9423 2744 1339 10139 2332 8771 3079 4312 7098 11256 1681 642 411 5179 11964 5793 8376 2386 9500 2401 5669 10501 1939 11311 4977 7401 8266 12472 480 10947 12116 539 7591 1020 1493 9017 2513 3100 4405 5679 3373 3795 10805 11445 10653 5898 5556 12139 12448 8448 5245 12533 10039 1324 2498 9955 6104 5516 204: 3474 7088 4085 10331 6972 7065 2023 10909 5915 5913 6491 5970 6936 5920 5919 5966 5944 5738 5968 6663 1233 5947 2258 10694 9592 4692 12344 11227 6753 8618 205: 9144 6127 6445 4401 3645 9756 5274 8302 1548 9875 9979 1922 1941 9100 7274 12121 11051 11528 9523 7830 3543 6760 1979 3997 9779 9635 4955 1818 946 5201 12580 8270 10531 415 4910 1802 2256 2979 7899 3139 3777 10332 10536 4842 8280 9000 1327 10950 8576 513 4263 6884 8684 3877 7243 7262 6420 1424 2680 10546 9965 11711 6656 1164 1160 5248 4812 11605 3598 8386 12446 3922 10305 467 5963 9481 1998 4655 4064 446 6112 6111 4689 3743 449 1123 11231 1143 42456 471 11629 6249 2152 2171 6494 8636 11953 5487 7844 6164 11566 1495 4623 6920 3447 3181 3153 1081 11890 3476 1127 1195 1192 12349 3600 11090 5377 8022 7160 11091 10643 7586 12247 6202 6217 4617 2237 2380 6219 1756 7456 925 3237 206: 5804 12016 10678 10712 10735 7448 9024 10738 10708 12014 10638 7423 7421 7417 309 10586 10603 10589 10584 7444 10644 7446 5047 10645 10646 12280 207 9710 2096 11839 9709 1612 8993 10037 6780 11613 9034 306 307 2004 11103 8166 6931 7311 6922 8933 10494 3783 308 11857 12034 3781 916 6666 9745 9140 6285 12449 10356 9452 4275 12246 9728 9405 2987 7223 2067 3934 8138 11430 9052 12318 6252 410 2407 6792 3564 2073 4786 11326 9877 3397 310 11058 9105 8474 12047 6860 7715 860 8446 4050 6973 6725 9408 4088 3842 1902 4332 2342 1701 10402 11870 4672 3986 10725 12181 1973 3950 9992 4578 10224 862 7045 11785 4789 5465 8088 3553 10189 9964 2793 6677 10001 3375 4200 10391 1361 1234 10741 10641 10683 11712 10743 10575 10581 4747 207: 12016 7448 12014 7423 309 5047 9710 2096 11839 1612 8993 10037 6780 9034 307 306 2004 6931 6922 8933 10494 308 11857 3781 916 9745 6666 9140 6285 12449 9452 8035 10356 11492 12021 4443 10064 8344 2067 3934 4275 8138 12246 9405 2987 9728 11430 9052 12318 6252 7223 410 6792 3564 2073 4786 9877 3397 310 11058 8474 9105 11870 12047 4672 3986 6860 7715 860 2342 8446 1701 4050 10402 6973 9408 4088 3842 4332 6725 1902 10725 12181 6779 1973 2823 9849 10154 862 7045 11785 4789 5465 10224 8088 4578 10189 3553 2793 6677 10001 3375 4200 10391 3950 9992 1361 1468 9964 7410 2176 10741 11712 10743 10575 10581 206 208: 8564 10720 7580 12251 9922 5975 8617 4257 645 3210 4615 8228 747 1408 10412 3357 4397 7547 10137 3018 7289 11413 1687 2058 4738 1274 12252 8769 6626 4708 2751 1442 2843 10230 6198 10814 2304 9207 209: 9386 8213 8184 6094 8240 8242 8209 8211 5327 9254 10652 9428 11965 11812 11814 9275 9274 6208 8173 7971 9276 9278 9280 9297 9253 8100 9330 9303 9305 4986 4730 10770 11755 3994 5070 7569 5734 3989 3985 9531 9214 9429 9365 11108 6372 5373 2117 3351 12521 4075 1896 3535 10982 4340 2371 858 3813 10602 5493 5548 10627 5552 2460 4278 1787 3297 2964 2965 2962 3630 9434 3625 4592 10087 8272 3870 4415 8484 5940 10629 10623 10636 10174 10667 5553 10670 10671 2562 2568 8456 5226 5200 11493 7169 9374 7962 11722 5462 2866 10150 10170 10153 4425 1856 4727 9772 6514 2550 9367 4482 9458 9455 2869 2162 9300 9302 10632 3616 210: 2857 3612 6601 1183 1181 1182 6604 1159 10118 10806 11819 11745 6639 11715 7049 10888 10024 7122 8076 8876 8903 1266 10535 624 7532 4011 5266 6168 6326 11178 2641 2461 6646 8758 7990 9318 8505 7393 2727 6008 3940 9115 5137 9096 1148 1363 10193 9377 9250 5445 11200 11273 11276 211: 11176 8570 11245 10274 6081 7181 6450 4624 9320 6129 984 7196 7388 2804 542 11805 212: 23933 4071 1789 8124 2340 3714 1395 1433 12303 375 2814 6364 9438 3292 12390 2984 6746 9695 675 2101 3618 12081 6128 1892 3448 9864 6152 2844 7381 4291 4973 5447 10140 11877 8566 7624 6472 10665 2089 9925 938 8536 6156 10608 11433 5967 1511 11974 12573 4734 11501 5076 12428 8275 2769 4402 11854 213: 4784 5997 2399 6338 3933 4092 10151 2740 10610 214: 10855 2954 6766 2958 8910 12101 6783 3620 7658 7785 3180 9266 9246 9247 1792 8649 5777 10173 10178 3461 9046 5810 5806 1226 3287 12557 8375 12235 8403 8384 7414 5429 4396 6501 8433 7094 8413 7920 5588 9853 6890 6483 9273 9841 683 9313 6871 6899 6877 2491 4890 9129 5744 9572 11085 12037 11048 12113 613 9424 6574 12066 7504 5863 8409 4273 10572 5923 1895 1893 9040 3665 5481 7755 8408 924 1454 12140 8378 5510 5509 5513 3124 3103 11911 4141 2082 2247 4630 8299 6667 702 8975 6801 745 741 779 770 772 744 771 11549 719 7117 5565 11875 215: 11919 9154 5594 10308 2827 2830 3408 3403 2471 5367 1120 5371 5081 4880 10931 7367 6883 11808 6136 2549 11638 6868 8315 3118 10508 10877 650 5616 4115 3026 3028 9516 785 9083 7596 8108 4176 6525 5765 3802 1806 8081 7208 8893 12007 8654 9048 9072 8575 8423 6300 6409 4165 6095 9477 2485 10112 5117 2278 2281 2264 2284 6055 2348 4251 8187 10826 9660 9216 2777 4403 7239 2643 782 2262 8111 1799 781 2696 8265 821 6575 9029 6259 5907 2153 9132 1008 9697 11658 5996 6135 3512 216: 1063 9995 9748 8083 4921 10081 2976 7153 8380 1072 2845 2124 5604 2742 217: 1063 9995 9748 8083 4921 10081 2976 7153 8380 1072 2845 2124 5604 2742 218: 10265 3604 11692 2087 2100 2084 4972 8627 4940 10555 4941 652 1430 11778 7581 915 1478 8934 1244 9538 6106 9540 6923 5854 6892 9462 3486 10996 12018 9346 3284 6742 8247 219: 5171 3451 10952 6452 5333 11383 12420 9816 9099 11249 528 11871 11060 8935 3521 3063 10253 9510 10954 6303 6941 523 904 5364 4534 1993 9623 3245 12506 8843 10612 7200 2319 7201 1746 9164 1043 220: 6376 1316 5391 12526 7194 2996 3154 10569 11756 11824 3924 9004 5150 5993 10023 5309 10233 5582 9183 5649 2780 11917 6719 11145 10056 2516 1372 5622 7269 2665 1402 5885 7636 6193 3223 2719 6657 1867 7660 12334 9360 5492 2710 2076 8465 7571 11887 2033 8847 3260 10323 11018 7553 6905 5747 10773 5018 9023 9420 9484 9512 8291 2650 4553 2233 4983 7834 11916 8565 4123 1090 3981 610 2885 5427 3349 649 9974 10523 10337 5840 8815 6996 11041 1321 11532 11331 9757 6755 2327 2730 5199 5280 11943 3656 6297 4570 2983 6557 12145 2376 7618 6924 9049 10975 8678 12452 7263 2204 3741 7210 7502 4325 11408 1350 6089 2892 8054 8643 2501 1647 11693 6378 1729 6966 8734 9027 8827 5647 9075 7286 659 4113 6496 4454 11650 4378 2224 2687 1763 830 3255 5001 3830 6495 3121 1757 7740 8530 2770 1866 459 2049 4814 12517 2408 8583 6850 7550 5545 2042 3709 5474 11062 4761 10345 7778 1449 1562 8901 4943 10916 11403 6820 3167 1997 7484 9833 12022 8573 5100 5639 7158 8791 9723 4484 10282 1334 11312 11317 4294 9400 4982 7125 2655 10854 9131 992 5153 2528 12519 12187 6818 799 861 11120 11361 6634 12230 10852 8817 3105 9513 8235 221: 6205 11358 3072 2888 2907 6203 2800 7221 4750 3627 12485 2816 10896 4463 3774 8273 5002 4122 8581 8364 3273 6044 6503 6451 6887 4226 5120 9987 679 12019 1695 939 9726 8964 2326 6178 6080 8551 12220 926 10271 3458 983 6773 5354 551 12326 1673 10474 7111 503 3261 7427 7498 5710 9522 12089 8842 8147 8799 9369 2355 6063 3582 3537 3557 1618 2519 10121 9781 10031 1438 4529 11657 7069 3979 1260 8752 9515 1762 10093 875 12460 12052 9166 7493 12523 10742 10451 8622 8931 10210 7668 3177 3657 6276 625 6423 222: 9766 2574 8653 12518 6881 10011 1281 4435 3555 696 5489 10478 6961 6001 1591 1453 10635 2267 6727 12366 4551 1889 1367 1388 9264 8099 5016 1033 4094 12546 7145 6511 1331 1524 3894 1943 8569 11313 5235 223: 12210 2632 5689 5995 9108 6848 3162 5357 9825 6099 9769 11406 12011 4089 11037 2154 7634 2930 6937 224: 11851 9599 392 3514 5363 9918 7949 12550 981 8255 3499 2997 9043 6076 2056 2922 11064 11131 9209 5316 10222 11118 1947 4743 225: 12336 2351 3767 1826 226: 9174 12242 516 9436 5692 6101 8462 9960 3910 227: 882 7014 8781 8246 10705 2703 8520 6497 7900 6599 3575 3216 228: 2359 5356 6318 6123 588 7908 6312 4748 9929 6824 6509 229: 9744 8168 1420 6853 7687 2503 7653 5252 787 6057 2759 8114 9054 8122 8127 4410 5238 4675 7892 11484 12365 11744 3437 6705 3241 11187 230: 9581 9205 10794 5085 2201 1384 4000 8605 4670 1972 10286 11576 686 913 2768 203 393 4753 1339 2332 8771 9423 7426 2744 3079 10139 4312 11256 7098 642 1681 411 11964 5179 1324 9955 6104 5516 9644 11807 7457 231: 12356 4958 6943 8532 8516 9081 4754 8450 8451 10677 4939 12575 11787 7205 4213 972 3291 9604 11517 7192 10860 5598 12538 4035 11116 695 7007 479 4154 10733 232: 641 10835 7416 7705 8597 5506 5365 2998 4911 1710 4507 7519 1965 233: 7211 12486 7508 11321 5086 11818 8707 9321 1682 4612 3885 10374 3698 2956 2709 2789 9060 3654 4690 9089 7726 3369 8385 2927 2192 5052 11202 11758 10190 5874 8038 8631 537 10655 4768 2120 3687 4281 11320 6521 4769 7545 7786 7407 12108 9206 12454 2147 7282 12432 3610 8128 5956 3069 234: 9373 9421 11561 11557 12294 10301 9284 6616 1308 6809 3915 11093 3919 11088 11597 11298 11592 6281 3917 11137 1726 11130 1230 3689 4740 3725 11047 2975 6172 1216 3544 4142 7375 746 11962 6474 12427 235: 9373 9421 11561 11557 12294 10301 9284 6616 1308 6809 3915 11093 3919 11088 11597 11298 11592 6281 3917 11137 1726 11130 1230 3689 4740 3725 11047 2975 6172 1216 3544 4142 7375 746 11962 6474 12427 236: 444 10758 1559 12502 889 9874 9788 7310 12020 6831 7980 10109 5949 6731 11689 7825 3697 1264 4393 548 2268 1773 3208 1147 4029 9056 1141 7469 5188 10443 7314 1452 1744 5383 237: 5650 5881 10697 3343 2506 6706 9195 3119 609 11113 12263 12264 9501 8410 8925 3221 7983 7956 933 2361 8269 9921 6336 10563 632 12541 10155 10751 9511 7976 6351 5482 10797 4571 1776 12112 7190 1900 9324 6339 7001 2317 9820 7015 6384 4917 11822 4227 11377 6229 10949 11498 1448 8172 10908 7776 6183 238: 3651 11823 2950 1915 5176 4381 8742 6316 9780 3427 8319 899 4829 11372 12232 6415 3788 1658 9838 11020 8918 7485 10102 8428 1054 2552 11363 12489 9487 10566 9535 11344 4210 1739 5067 8368 9789 7897 2937 10388 8859 10675 3146 1783 2989 3471 4847 919 918 5832 1172 2121 5023 806 11459 12478 12285 11359 2683 11412 12180 11214 5716 7022 8289 6594 7858 11270 1848 12273 9776 6464 1578 4239 7235 5329 9074 3608 6048 1812 3310 7872 5540 8662 4796 790 2336 6532 8866 6741 7383 5683 4201 1638 1583 6819 11937 2788 11593 12298 6125 6977 1956 8141 7002 1569 11618 3937 5648 10925 10480 9137 6221 2366 6277 10503 5161 12302 5628 4791 239: 3651 11823 2950 1915 5176 4381 8742 6316 9780 3427 8319 899 4829 11372 12232 6415 3788 1658 9838 11020 8918 7485 10102 8428 1054 2552 11363 12489 9487 10566 9535 11344 4210 1739 5067 8368 9789 7897 2937 10388 8859 10675 3146 1783 2989 3471 4847 919 918 5832 1172 2121 5023 806 11459 12478 12285 11359 2683 11412 12180 11214 5716 7022 8289 6594 7858 11270 1848 12273 9776 6464 1578 4239 7235 5329 9074 3608 6048 1812 3310 7872 5540 8662 4796 790 2336 6532 8866 6741 7383 5683 4201 1638 1583 6819 11937 2788 11593 12298 6125 6977 1956 8141 7002 1569 11618 3937 5648 10925 10480 9137 6221 2366 6277 10503 5161 12302 5628 4791 240: 5298 3673 6171 5229 8230 6271 9427 1356 10882 11852 10687 6088 10076 9830 10597 6373 3987 10322 241: 2393 3407 11789 11391 11346 5568 689 9121 3768 6558 5447 1870 7849 2504 8733 10066 994 11743 980 4909 7933 8486 8369 5152 1705 6156 10608 242: 3431 5395 4346 8330 8327 8702 7787 5265 8943 12561 4536 11625 4411 1035 11796 6078 2720 4449 10010 3057 9876 3536 5603 11727 5025 698 9899 6457 10804 3454 2741 11343 11668 12537 9198 9194 6906 11749 2886 4118 11050 3125 3104 8238 7647 11157 11552 5735 3190 1224 2010 10669 3186 12278 10534 9546 10088 3888 1521 10626 10413 11620 12324 1406 12498 3067 7386 6359 10120 6004 2803 9290 11141 854 2391 6032 10433 12371 11636 11795 6713 8567 10754 717 2465 9545 9886 6990 4012 8324 3742 1053 8586 8683 10073 12149 7481 2755 2646 6082 8956 4440 4579 4447 6886 4268 561 11512 3439 1568 8328 1091 7948 5861 726 582 11893 2118 12271 6845 6843 6847 2068 9119 2022 8587 12175 8754 6777 497 9325 6872 7531 11335 2928 9885 5358 1963 6109 7533 11337 11444 9889 6179 1632 6874 3342 12072 12199 11476 9892 6224 6254 5355 11338 7625 10426 10428 11395 1315 11066 11063 11076 5351 1440 11336 11316 3538 8749 9778 5154 9356 835 831 2589 8503 10727 2891 8958 11046 6790 9818 9094 9828 5374 8610 7368 244: 9283 4282 1754 571 9388 10252 4060 4063 10254 9392 10256 10251 8049 10250 10237 10240 8942 10188 9163 9457 9412 4148 2795 2440 10370 3577 434 12383 7841 4935 4928 4931 4933 4908 701 7640 2602 8966 4824 5455 4822 4823 5454 9020 8692 9551 6293 8301 3456 1197 12421 3247 6475 7319 9389 569 6169 1497 1499 9460 2309 600 2172 2178 6997 2668 8309 2821 4458 2940 9832 5380 6918 732 9375 9376 9294 2109 12129 8117 12379 4965 10550 7218 12182 11653 9067 4197 9956 4905 3379 8543 5663 10834 8946 3814 4643 1788 6732 9857 4189 10740 6347 1240 5417 6399 1781 1782 3068 3398 10951 3402 4557 3406 1307 4161 11960 8337 8307 7812 2486 2510 1431 6430 894 5282 12422 6029 10938 10935 10936 2671 4260 10891 7914 5646 9237 11207 11111 4869 10685 6067 459612455 4572 7777 2072 1364 3840 4879 1193 7010 1667 2748 4809 1850 8067 8922 9994 5724 12160 6849 3432 11503 11999 2619 11453 5074 12050 3193 3298 5344 3303 3301 3300 3362 3283 3251 3211 9607 3277 4512 10924 8703 8838 4726 6467 2527 962 5203 4589 9809 9806 9805 9812 1242 312314 3674 3728 3676 4145 7690 4132 3000 3002 2009 2415 3348 2543 1825 3368 1753 11838 6270 11163 4736 11353 3911 10235 1023 29385 10283 10275 10280 10279 1285 1293 1279 1312 1291 1089 2708 1407 10065 9873 12041 9138 11097 4104 405 10732 3843 10734 9232 1527 5586 4841 245: 9283 4282 1754 571 9388 10252 4060 4063 10254 9392 1025610251 8049 10250 10237 10240 8942 10188 9163 9457 9412 4148 2795 2440 10370 3577 434 12383 7841 4935 4928 4931 4933 4908 701 7640 2602 8966 4824 5455 4822 4823 5454 9020 8692 9551 6293 8301 3456 1197 12421 3247 6475 7319 9389 569 6169 1497 1499 9460 2309 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7074 1179 12458 1855 12015 9980 10716 10918 11770 10255 581 10006 4467 10169 7675 1250 5319 9827 496 1732 8778 247: 9530 12217 826 5267 2006 547 7365 1816 10509 6784 2509 7339 3841 5471 11850 11519 2806 7443 8018 11751 1529 4077 12088 6149 1489 868 7473 2466 614 1176 6038 2195 1557 8182 3984 248: 2809 1347 5519 6353 8932 7422 7557 4683 11147 10637 9404 11768 910 8870 6324 5045 4945 9453 7164 4152 3322 10499 7328 8994 11460 9634 9064 9047 10906 11904 749 5014 9906 9939 9963 2364 6617 1635 4234 3462 12043 10075 3459 10489 8606 1999 4508 10028 5341 6180 9971 7215 3422 5091 9652 10601 8028 11427 6678 4365 7082 1022 3209 4357 9159 7321 12077 11967 680 7943 661 11246 10622 7676 818 3364 9160 6187 8531 4604 6381 4859 2298 10753 12368 9200 5155 2175 8058 11106 1639 5299 1905 1068 110937 1738 3580 7344 8320 8466 5687 5293 8844 5030 1572 1735 543 1105 562 3483 9147 12109 11282 7291 10666 11009 2523 12408 12128 12406 2535 10560 12212 10101 11610 5148 8687 5945 11651 12286 5942 12291 11624 11675 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12482 7571 11887 6428 3260 11018 10323 834 11437 5486 604 7553 388 11872 9188 4081 6905 11306 7202 8348 5018 9023 9484 9512 4553 11385 2233 1463 8789 7834 11916 4286 2350 11883 8565 1749 1023 10744 4181 6476 5850 1090 11086 5178 2911 2349 5771 610 2885 5427 3735 3349 649 847 11740 439 4459 10523 846 4315 7823 8415 6975 9073 10143 8957 2315 2292 6603 1321 8167 11331 9757 6755 2327 2730 11943 11848 5411 5103 10812 8103 4922 9555 12323 9526 3046 4303 9846 9636 10415 2376 7618 90491 18631 2452 9813 8070 10257 3030 4031 4065 17161 2281 6543 5890 6492 3741 8608 6220 3053 9086 637 6653 8593 11408 12357 6247 4629 2842 5476 10303 2032 8190 1284 12440 9307 3477 2901 4407 5191 10111 4218 4412 11723 7627 1891 7522 2323 8023 4766 8763 3951 8643 2501 5759 1647 1794 11762 5216 5233 8857 11939 11126 5668 5621 11780 2443 5526 448 7298 6966 8734 1104 5647 2321 12229 10556 1362 7312 5089 4558 784 7286 2999 10921 10659 6804 659 6020 7233 6496 4454 10299 11650 6596 4378 2529 10338 1375 573 2224 12213 1763 1605 830 5001 10446 3830 4701 776 9314 6716 7514 3121 8862 3508 8463 1757 6913 8530 5097 7740 2770 459 4180 2049 3855 9103 6909 2972 4374 2939 12517 6215 6073 6945 11644 2408 8583 12484 6919 5474 3709 3313 12163 3116 9475 8417 10673 6369 4831 1516 1241 7797 6820 8998 1997 1768 2328 7424 11978 8750 7780 11894 3142 12577 12293 11312 2772 11537 10690 1718 8646 8086 4294 9402 9400 4982 4149 8342 12024 2655 10289 6995 8495 7186 9136 9600 4718 3389 4542 519 4041 992 7372 1002 11226 2528 12519 1542 11389 1514 6818 3426 4183 9065 7662 8073 521 1525 6163 861 8690 2566 11361 9685 12230 8817 4888 354: 5626 2966 4663 4805 5637 8787 12445 11680 355: 9746 2454 2820 6126 9527 11327 4206 1124 356: 3803 2159 11265 10262 1208 2265 6548 4559 9949 8892 1253 5869 6212 6017 11175 3155 5749 2919 12006 2161 7594 10558 12270 357: 11897 12310 11976 5165 7935 565 11277 908 12483 7089 11639 358: 7374 4134 5499 4481 11052 4793 5466 6747 3954 6213 640 4867 12055 11571 3592 1079 5345 10097 11174 7895 359: 10455 9279 2524 4394 10985 4178 3240 6391 10552 3567 2029 1042 1797 11402 7449 8268 2462 865 5988 3908 14041 1416 5916 7278 7325 12549 7885 7883 6455 11569 6302 12413 9181 1962 2013 3143 8399 10462 7807 7806 3136 3131 11003 1251 7387 2412 10649 10077 4839 7683 6752 8353 3902 11971 9700 7909 1974 1145 11946 8178 8457 360: 243 8836 3558 6739 5558 11923 11784 11730 10397 3528 7134 8772 7228 2453 8340 7839 1256 10038 12184 361: 10213 10215 12148 10197 1692 10198 10202 12341 3385 2836 6484 11717 10618 3411 10927 6460 1921 10241 1907 12389 7827 9025 5807 6681 12391 12411 6550 974 5415 9578 5213 4987 4080 12522 2369 12124 4767 6352 7944 10217 10216 3413 9071 3511 11013 10218 7981 8298 9862 2132 530 3844 6662 6625 4550 6341 1722 7027 9323 5094 3611 2266 4530 7763 7290 11753 2081 4242 11360 1239 2673 4531 8677 4930 2570 7994 6309 4476 9282 6794 3851 3634 3637 10242 10245 362: 922 10181 10184 6562 3258 6528 6629 1180 9451 406 6910 5922 8885 9557 3599 7579 7047 2882 10405 3975 532 1868 2867 574 1859 6925 6880 950 10421 6722 1464 1500 5038 12068 3134 12241 9985 5705 3554 11752 2337 1748 4287 7838 8011 5651 5941 9051 5564 4621 7863 2536 5059 5773 5837 12335 6680 7099 7118 10998 2432 2463 1750 9628 6813 3628 9033 5836 10449 5420 5207 3387 7412 3524 394 11949 8547 4036 6873 1600 1443 5349 4233 9783 7318 2864 11057 1323 3052 10007 2599 6329 9359 7436 7657 11879 1025 12197 9682 10707 9968 4445 801 8656 6142 2948 7184 4255 10302 10364 12386 73091 2216 6438 2050 3050 2920 5965 11072 9831 3854 5662 12003 3011 2728 9738 1017 8134 526 363: 6176 8639 1152 1522 8352 6375 10201 5139 8072 2308 4150 3445 7428 2024 4633 4866 2581 12337 12476 10035 8153 11036 3036 7174 5414 3759 9595 1795 7561 5236 11547 1813 1163 4779 3695 4760 6556 8661 11299 11986 8914 10050 11070 4068 809 4568 3229 10263 4659 10993 8382 1047 2931 9895 4478 1570 7237 4269 3838 1016 4137 11183 12276 11482 9854 11264 3433 7046 8216 3513 9383 8148 1602 6058 2215 1842 5999 10212 8258 6591 4686 9520 8497 6648 3088 6786 5986 4715 7335 6510 1839 5658 2103 11352 7577 4634 7828 9426 11791 11970 560 12269 10454 3529 7253 5891 880 4009 2760 7540 9015 8638 3356 5775 10574 1338 12117 3249 7644 9762 3751 7042 3672 4202 5589 6019 9835 7229 7693 3086 10296 9608 11707 2588 6425 9954 7071 10204 7525 6469 3192 5805 6230 4385 2180 6991 11841 9662 5353 2773 10791 3059 6304 7745 5886 6518 1536 1863 1385 5022 6003 8904 7257 9755 11607 11221 6807 4696 8329 9798 1221 364: 4714 6552 6036 9637 11177 7157 2425 8841 4455 8468 3444 8404 3936 763 10942 1668 885 1786 10802 7271 8055 4969 11623 3054 11828 2575 5685 5924 5460 4819 9989 11834 10380 5520 9589 3334 4912 9894 7974 8700 12342 365: 4543 9647 3507 1211 3246 10549 2019 2094 4177 4338 9238 5770 4772 6343 4048 12231 10905 12008 4926 12442 2270 5780 11506 7565 1094 12147 1451 473 3294 11314 9173 2035 2593 12094 5494 5488 3087 2026 8206 9878 4705 10472 4746 6642 5937 8790 11896 2244 5026 5835 7953 11936 10110 7133 3785 366: 10341 5696 6298 2325 2938 8027 1580 7429 589 12057 10353 3976 3860 1646 9225 842 5818 4573 1901 8803 367: 4208 3058 2394 8507 2917 6904 1345 10919 4336 1202 7187 1114 3353 3660 2387 8437 9496 9413 11082 8079 12040 2841 4934 12221 8040 4875 7674 11844 10774 6033 11664 5283 5192 1085 3967 2564 3007 11334 11393 9940 872 2508 12169 3721 6130 1306 5401 10879 883 9814 5802 10180 6895 7491 3952 4386 8159 1106 5271 6278 4124 11612 6546 556 9556 5366 11798 6295 10902 3531 3572 3574 7418 3765 2343 7356 6349 9808 3624 9583 6431 3128 8098 3871 2494 1319 2271 4787 1838 1456 5064 6878 9972 8908 6774 3809 8730 11094 670 1496 5212 3588 9459 8518 6963 10018 7791 8264 2418 6442 11211 1636 6014 5278 4155 11100 10528 11481 10313 7126 8887 2600 11160 3425 5846 4898 4527 6393 12267 1747 10239 10330 3670 10329 10327 11203 10715 8883 10249 1046 11155 5228 2070 2128 7076 6412 6875 5853 8795 564 9262 2640 5083 1875 6898 6951 12373 7127 368: 6754 10585 6689 9826 964 4693 8822 6462 691 2410 9712 9884 3219 7232 12539 1991 11008 9322 8725 369: 1461 7305 10596 7966 8323 9036 4856 433 2663 8335 8509 11775 10231 5820 8056 12556 5630 11866 9292 2220 509 12153 7303 12393 7926 370: 1200 12300 576 5306 8372 2320 7732 6788 3633 10013 657 1403 10074 7663 6585 8051 2569 2722 8325 1121 11616 4248 6146 4276 2647 5766 9625 10767 6273 4186 6188 3988 4877 3998 8151 6018 2040 595 371: 1200 12300 6283 5306 1573 8372 9432 1142 533 2320 7732 370 1267 595 3852 372: 7477 1676 4448 2400 6045 6940 9923 11995 8913 10513 634 7969 11746 6446 4371 1018 4026 10874 11604 5505 9219 4140 6427 12025 1669 3605 5027 10847 11128 1483 9362 2044 12343 3560 430 11220 12146 10479 7769 4704 5385 12424 3286 1435 12461 10990 7985 5652 6086 12067 2785 6367 3470 2598 10591 4756 2959 10900 11878 12536 5795 12559 5450 8391 10046 7073 3157 2834 8144 2236 2782 1926 9617 1987 2822 2279 2196 10312 5369 10124 11903 12289 4170 12092 8540 2362 12010 5204 9431 6620 3500 9585 7989 1508 4885 877 6119 3807 2585 12330 4380 3820 5302 3056 6337 2060 3096 202 4492 1604 11737 710 8574 11646 3171 9030 765 5680 10955 6592 2624 9114 5576 5196 5912 1980 11275 1119 3929 9810 12099 11930 4546 3504 1950 7696 374 1703 373: 7477 1676 4448 2400 6045 6940 9923 8913 10513 6341 1746 6446 4371 1018 4026 11604 5505 9219 4140 6427 12025 3605 11128 9362 1483 5027 10847 2044 12343 3560 430 11220 12146 12424 3286 12461 10479 10990 5652 5385 6086 4704 7769 7985 2785 6367 10591 10900 11878 12536 12559 12067 10046 3157 5450 8144 9617 1987 2822 10312 2196 5369 10124 11903 12289 2362 12010 6620 5204 7989 4885 877 4170 2585 8540 4380 12092 3820 1508 3056 6337 5302 3096 2060 202 4492 1604 11737 710 8574 11646 3171 9030 765 5680 2624 9114 6592 10955 5196 5912 1980 11275 9810 12099 4821 10994 8060 1531 6262 4632 1058 2716 7317 4825 3731 5786 414 5576 1000 9714 1854 2489 4638 4485 11930 3504 1950 7696 374 372 1703 374: 7477 1676 4448 2400 6045 6940 9923 11995 8913 10513 634 7969 11746 6446 4371 1018 4026 10874 11604 5505 9219 4140 6427 12025 3605 1669 10847 5027 11128 9362 1483 2044 12343 3560 430 11220 12146 10479 4704 7769 5385 12424 3286 12461 10990 7985 5652 6086 1435 12067 2785 6367 3470 10591 2959 10900 2598 11878 12536 12559 5450 10046 7073 3157 2834 5795 8144 9617 10146 1987 2822 2279 2196 10312 5369 10124 11903 12289 4170 2362 12010 5204 6620 12092 8540 9585 9431 3500 7989 1508 4885 877 2585 4380 3820 3056 6337 5302 2060 3096 202 4492 1604 11737 710 8574 11646 3171 9030 765 5680 6592 10955 2624 9114 5196 5912 1980 11275 5576 9810 12099 11930 4546 3504 1950 7696 372 375: 2393 3407 11789 2984 10140 7809 9834 6717 6156 10608 6364 376: 6589 11497 6836 11332 11382 5877 2213 2951 8215 1064 1057 8227 8249 10551 3319 8195 8218 8221 9142 9661 8198 6116 10889 10520 7399 3659 1734 5077 3510 5010 8351 9403 9244 483 4466 12167 7031 4216 6477 4107 12150 6828 5305 12404 1567 7248 11614 7445 3482 5079 2734 11033 3311 7402 4221 2223 1040 9289 2902 4438 4600 4324 9168 2576 10382 7057 4976 1719 10894 10483 3361 9016 5634 7496 1784 9760 12134 7188 8533 10833 1303 9336 4810 10290 3800 11192 6040 852 586 9907 11699 8192 7242 2963 10418 12069 9588 10604 2330 9242 6800 10782 7530 6261 8220 8962 7198 7306 7697 2102 2085 9372 5757 3700 11432 3755 11635 3376 11831 9573 10934 12553 9010 12441 2757 3093 11913 9725 3144 5892 10167 7130 5337 3872 1625 4143 7507 3179 7982 2590 1203 1932 1225 6260 1044 9032 498 499 9053 7023 8322 4927 3331 10393 8132 11728 8655 7771 988 8909 1791 5413 2157 10976 1365 2511 3200 7167 9986 11603 1041 6238 10166 11045 9704 9621 7669 2014 3281 11206 2697 4067 5118 12401 5368 12284 9430 7136 12412 6387 9543 2522 572 5119 1606 8394 10248 11146 10285 11719 6776 11309 11139 10439 9245 11197 11729 2036 4759 9773 6077 5571 11810 10084 10857 8191 9597 8037 3279 8225 797 976 977 979 999 5185 4166 9598 4901 7135 2944 10328 8189 6781 377: 10527 6513 5978 2596 1034 5101 1637 6141 6160 5334 12110 378: 11873 5442 7939 7815 10258 11591 11595 4116 4129 9663 4114 11588 11586 11585 9690 11556 4111 11550 9687 4264 11554 5405 4130 3884 9667 3491 11545 3879 11546 3875 9646 3873 4105 3881 4101 4086 4084 3850 4082 4078 8588 8545 8589 9271 8300 5033 5035 5058 1832 5005 9380 5056 2097 1720 7704 10811 2733 5138 3490 8251 3805 2314 3573 5858 2753 2629 7295 557 3786 8599 9153 2012 5249 12190 6141 5334 6162 7529 1731 10108 463 12105 5048 660 7731 10191 9632 11189 6866 10706 4292 7977 437 10106 5124 1552 2131 7651 11225 8644 1923 3585 6432 3532 379: 8061 8064 7961 7964 404 3270 11215 3265 10192 11241 2078 11168 7871 12087 4499 1544 1412 6268 8820 11677 914 5507 4996 4991 4416 4419 1981 4347 3401 10092 11767 11031 12403 9799 8861 3101 12558 10974 1155 1157 1162 1171 1167 10984 5609 5612 5156 5809 5813 2069 10182 4042 3226 7512 6791 5053 6674 11820 11674 10970 9466 11069 10506 2468 2442 2444 2291 2435 2288 2294 2296 1196 12392 12414 12417 12418 12430 12435 12437 12453 8217 3323 4516 10500 12038 2221 5834 3035 7400 2413 1207 2833 2850 4552 4556 3578 5581 555 7183 552 7176 553 7179 549 7185 4518 4522 4539 4541 3889 12474 5841 9042 5812 8199 8200 8494 469 4590 6355 711 9845 9847 4984 7919 7432 7433 4285 11121 4503 7100 5439 2561 5559 1944 1237 10062 2402 4735 6583 3891 5718 5725 5746 9737 2946 2949 7280 5538 2658 2674 9057 8712 9135 5144 6967 11626 10759 9277 11254 11258 10005 400 1615 4506 2137 6636 12186 843 8021 3464 4335 12045 3009 6698 7468 1733 3239 4185 12228 1011 4131 5279 11790 5453 5452 9366 10461 6840 5340 4790 9069 4421 767 6182 2807 11421 6683 6090 4254 11473 4256 7225 11457 6361 5623 1560 2751 6147 6148 6151 11409 11411 11415 9097 1310 3111 4301 9508 7650 1796 11061 11104 11934 6530 9063 10105 4959 7479 8137 11271 4864 11951 8624 8110 5704 4119 6365 9492 7963 10912 2447 3204 3317 2521 2451 2450 2546 2514 3202 2487 2365 3205 2363 3148 2396 3354 2480 4047 3352 3203 3175 3243 2484 3262 3173 2419 2335 3380 3316 4051 3164 9189 11975 11977 12013 7351 6499 6517 11125 10880 9640 9651 5762 5769 11996 11736 6987 8226 3336 8224 1210 722 7083 11418 5972 9384 11424 6600 7623 7528 10940 10803 8430 3662 3099 4359 5542 1686 11972 8902 2017 2110 6669 6692 11541 4284 1473 4147 8879 8882 4376 8535 9364 10522 7701 8652 11989 10456 4561 12185 1501 3769 9669 5220 5218 9616 2405 12056 491 492 8338 819 2222 6325 1906 9158 6199 421 5392 2403 3750 694 697 699 724 728 6340 3693 3696 3955 7148 1166 2645 2409 8777 4990 8278 8279 8281 8304 8306 8308 8312 947 4453 1206 10609 10755 11735 11539 11786 1546 10196 7632 3082 3037 3110 3106 6524 3289 12083 12085 12082 490 6197 12080 12079 2219 8066 6405 8894 7333 6385 10897 8930 1223 3912 11910 1912 8891 3475 4698 2662 901 1194 1503 1507 8696 9203 6471 566 6133 3248 11892 5459 3302 8553 4703 8621 851 4337 4341 3675 2970 5601 10607 9524 12425 7307 9223 3918 736 729 961 10464 6196 1543 2758 2971 5700 12211 2578 10438 3880 1539 8865 8981 3738 12457 445 3540 11456 5703 4762 8680 2839 583 4627 8732 5599 2623 6321 5659 10004 10168 8741 6218 2194 2214 6554 6244 6555 2191 7472 8949 2896 6699 6703 3469 4902 9719 1995 5537 3957 3964 11679 3719 424 8848 3586 11832 7055 10785 9507 7643 10467 7642 10395 8808 9495 8628 9050 12192 9591 3681 7246 11257 10605 9721 2379 1187 1674 10009 1108 6522 3363 3360 1049 1051 1056 7756 7738 8897 2974 8924 2988 7438 832 4666 11859 4702 2991 4355 8867 4968 8868 3897 8895 2967 4669 4697 4674 4700 9415 6838 6549 2617 621 1831 10829 10808 4794 6627 4776 7605 8694 12200 11056 11002 7868 3699 2610 419 418 10435 936 2345 6762 12543 9549 10661 10689 10699 10719 1190 1165 1161 1170 11929 11419 7988 3798 1218 1220 2721 1800 6167 1268 3178 380: 7583 12223 3882 6192 10967 5287 11700 9299 12204 3071 9116 6233 3626 6939 5957 2854 2852 10080 6245 9582 11285 9334 2429 381: 611 12004 6814 5320 1380 5991 7802 5112 6551 402 7832 382 11138 382: 611 12004 5991 6551 7832 383: 4837 10247 4513 8974 9193 3250 11577 10663 2910 9363 1186 384: 11191 6118 591 9506 9370 8717 1055 9504 10360 6606 575 5878 5911 3404 1764 7501 4369 4367 12240 12238 2227 7439 2003 7437 7440 7521 11862 2334 12466 12137 5673 11590 12090 11766 7515 5121 3935 6239 6232 836 6307 6289 6328 6310 9157 8674 2257 2828 2851 6282 1990 10944 5126 5127 905 3876 907 8578 912 8514 8899 12309 12059 10972 11683 4862 4099 11900 9518 9521 4985 9517 6702 960 9819 958 965 963 2731 11043 10487 10486 11010 7682 1759 9618 9619 5901 10142 7649 753 755 4752 6402 11686 1413 656 5951 9563 795 8523 4826 11233 3115 385: 12282 3816 6979 4487 8194 4174 1027 3606 9593 8612 10293 4967 3304 6074 6908 8506 6658 7332 8682 9519 4399 4231 3446 3597 11520 11676 11673 11704 11678 11714 11703 5090 10122 4379 386: 3874 2573 7549 7517 9650 3332 1953 4431 11909 3497 10045 2551 2545 9861 8988 4509 11527 10422 4863 8961 11568 5627 2724 9699 5675 8512 2715 4144

Example 6

This example illustrates the preparation and identification by screening of transgenic seeds and plants having enhanced agronomic traits using DNA encoding homologs identified in Example 7. Transgenic corn, soybean or cotton seed and plants with recombinant DNA encoding each of the homologs identified in Example 5 are prepared by transformation. The transgenic seed, plantlets and progeny plants are screened for nitrogen use efficiency, yield, water use efficiency, growth under cold stress and seed composition change. Transgenic plants and seed having at least one enhanced agronomic trait of this invention are identified.

Example 7

This example illustrates the identification of consensus amino acid sequence for the proteins and homologs encoded by DNA that is used to prepare the transgenic seed and plants of this invention having enhanced agronomic traits.

ClustalW program was selected for multiple sequence alignments of the amino acid sequence of SEQ ID NO: 371 and 11 homologs. Three major factors affecting the sequence alignments dramatically are (1) protein weight matrices; (2) gap open penalty; (3) gap extension penalty. Protein weight matrices available for ClustalW program include Blosum, Pam and Gonnet series. Those parameters with gap open penalty and gap extension penalty were extensively tested. On the basis of the test results, Blosum weight matrix, gap open penalty of 10 and gap extension penalty of 1 were chosen for multiple sequence alignment. Attached are the sequences of SEQ ID NO: 371, its homologs and the consensus sequence at the end. The symbols for consensus sequence are (1) uppercase letters for 100% identity in all positions of multiple sequence alignment output; (2) lowercase letters for >=70% identity; symbol; (3) “X” indicated <70% identity; (4) dashes “-” meaning that gaps were in >=70% sequences.

SEQ ID NO: 371 MDIFDNSDLEYLVDEFH--ADFDDDEPFGEVDVTSESDSDFMDSDFDFELSESKTNNETS 12300 MDIFDNSDLEYLVDDFHGFSDSEDDEPFGEFDHKSEADSDFEDDLDPTQESD------TS 6283 MEHFNNDDLEYVVDEYYDVPDFAVEDTS---SDIVPELTSDVDSDFEDEFPTSNAKTDTT 1573 MEHFNNDDLEYVVDEYYDVPDFAVEDTS---SDIVPELTSDVDSDFEDEFPTSNAKTDTT 8372 MEHFNNDDLEYVVDEYYDVPDFAVEDTS---SDIVPELTSDVDSDFEDEFPTSNAKTDTT 5306 MEHFNNDDLEYVVDEYYDVPDFAVEDTS---SDIVPELTSDVDSDFEDEFPTSNAKTDTT 9432 ------------------------------------------------------------ 533 ------------------------------------------------------------ 2320 ------------------------------------------------------------ 1142 -------------------------------------------------MTISNTSSTSK 1200 ------------------------------------------------------------ 7732 -----------------MAHDLHDDLEFVSGDDDDYYLEFDHDPGHGFHTSAATSASQTL consensus xxxxxxxxxxxxxxxxxxxxxxxxxxxx---xxxxxxxxxxxxxxxxxxxxxxxxxxxxx ALEARNGKDIQGIPWESLNYTRDRYRENRLLHYKNFESLFRSREELDKECLQVEKGKNFY ALEARNGKDIQGIPWERLNYSRDQYRYKRLQQYKNFEILFRSRQDLDKECLQVEKGKHFY ASEARNGKDIQGIPWERLNYSRDKYRETRLKQYKNYQNFSRSRHDLRKECLEVQKGETFY ASEARNGKDIQGIPWERLNYSRDKYRETRLKQYKNYQNFSRSRHDLRKECLEVQKGETFY ASEARNGKDIQGIPWERLNYSRDKYRETRLKQYKNYQNFSRSRHDLRKECLEVQKGETFY ASEARNGKDIQGIPWERLNYSRDKYRETRLKQYKNYQNFSRSRHDLRKECLEVQKGETFY -----------GIPWERLNYSRDKYRETRLKQYKNYQNFSRSRHDLRKECFEVQKGETFY -----------GIPWERLNYSRDKYRETRLKQYKNYQNFSLSPHHLHKECFQVQKGQTFY -----------GIPWERLNYSRDKYRETRLKQYKNYQNFSRSPHHLRKECFQVQKGQTFY TIFRRNGKDIQGIPWERLNYSRDKYRETRLKQYKNYQNFSLSPHHLHKECFQVQKGQTFY ------------IPWERLQITRKDYRKARLEQYKNYENFPQSGELMDKLCKQVESSSKYY IGALYFRTSRWTIPWERLNYSRNQYREMRLRQYKNYENLTMPRDGLEKECKQVERKDTFY xxxxxxxxxxxgIPWErLnysRdxYRexRLxqYKNyxnfxxsxxxlxKeCxxVxkgxtfY DFQFNTRLVKSTIAHFQLR----------------NLVWATSKHDVYFMNNYSLMHWSSL DFQFNTRLVKSTIAHFQLR----------------NLLWATTKHDVYFMKNYSLMHWSSL DFFFNTRLVKSTIVHFQLR----------------NLLWATSKHDVYFMQNYSVMHWSAL DFFFNTRLVKSTIVHFQLLRQVXVSSLAGPNIMLRNLLWATSKNDVYFMQNYSVMHWSAL DFFFNTRLVRXTLAGPNIMLR--------------NLLWATSKHDVYFMQNYSVMHWSAL DFFFNTRLVKSTIVHFQLRPN----------IMLRNLLWATSKHDVYFMQNYSVNHWSAL DFFFNTRLVKSTIVHFQLR----------------NLLWATSKHDVYFMQNYSVMHWSAL DFFFNTRLVKSTIVHFQLRN----------------LLWATSKHDVYLMQNYSVMHWSAL DFFFNTRLVKSTIVHFQLQLGRTX-------IMLRNLLWATSKHDVYLMQNYSVMHWSAL DFFFNTRLVKSTIVHFQLLXRWNMSSLAGPYIMLRNLLWATSKHDVYLMQDYSVMHWSAL EFQYNTRIVKPSILHFQLR----------------NLLWATSKHDVYFMSNSTVGHWSSL DFHLNTRLVKSTTVHFQLR----------------NLLWATSKHDVYLMQNYSVMHWSSL dFxfNTRlVkstixhfglxxx----------xxxxnLlWATsKHDVYxMqnysvmHWSxL LQRGKEVLNVAKPIVPSMKQHGSLSQSVSRVQISTMAVKDDLKLREGSKESLSVRKSTNL LQRSKEVLNVAKPIVPTMKQPGLLSQSISRVQISTMAVKDDLIVAGGFQGELICKRINEP LRRGKEVLNVAKPIIPTLKRPGFLAQPVSRVQISTMTVKENLMVAGGFQGELICKNLKHP LRRGKEVLNVAKPIIPTLKRPGFLAQPVSRVQISTMTVKENLMVAGGX-SRVSLYNLKHP LRRGKEVLNVAKPIIPTLKRPGFLAQPVSRVQISTMTVKENLMVAGGFQGELICKNLKHP LRRGKEVLNVAKPIIPTLKRPGFLAQPVSRVQISTMTVKENLMVAGGFQGELICKNLKHP LRRGKEVLNVAKPIIPTLKRPGFLAQPVSRVQISTMTVKENLMVAGGFQGELICKNLKHP LQRSKEVLNVAKPIIPTLTHPGFLAQPVSRVQISTMTVKENLMVAGGFQGELICKNLKQP LRRGKEVLNVAKPIIPTLKRPGFLAQPVSRVQISTMTVKENLMVAGGFQGELICKNLKHP LQRSKEVLNVAKPIIPTLTHPGFLAQPVSRVQISTMTVKENLMVAGGFQGELICKVGLII SHKMTDVLDFSGHVAPAKKHPGCALEGFTGVQVSTLAVNEGLLVAGGFQGELVCKSLGER LQRGKEVLNVAGQLAPSQNVR--GAMPLSRVQISTMAVKGNLMVAGGFQGELICKYVDKP lxrxkeVLnvakpixptxkxpgxlaqpvsrVQiSTmxVkenLmvagGfqgelickxxxxp RLLSALN----------------------------------------------------- GVAFCTVLHRFX-NDITNSVDIYNAPSGSLRVITANNDCTVRVLDAXNFAFLNSFTL--- GVLFCGKITTDDNAITHAV-DVYSNPAGSLRVITANNDFQGRVFD--------------- GVLFCGKITTDDNAITNAV-DVYSNPAGSLRVITANNDFQVRVFDAENFASLGWFKYDWS GVLFCGKITTDDNAITNAV-DVYSNPAGSLRVITANNDFQVRVFDAENFASLGCFKYDWS GVLFCGKITTDDNAITNAV-DVYSNPAGSLRVITANNDFQVRVFDAENFASLGCFKYDWS GVLFCGKITTDDNAITNAV-DVYRNPAGSEGNPA-------------------------- GVLFCGKITTDGNAITNAVXDVYRNPAGSLRVITAXNDSQASGFDAENFAS--------- GVLFCGKITTDDNAITNAV-DVYSNPAGSLRVITANNDFQVRVFDAENFASLGCFKYDWS ISYFHSI----------------------------------------------------- DVKFCTRTTLSDNAITNAM-DIHRSTSGSLRITVSNNDSGVREFDMERFQLLNHFRFNWP GVAFCTNLTGNNNSITNAV-DIYQAPNGGTRITTANNDCVVRTFDTERFSLISHFAFPWS gvxfcxxxtxxxnxitxax-dxyxxpxgsxrxxxxxndxxxxxxdxxxxxxxxxxxxxxx ------------------------------------------------------------ ------------------------------------------------------------ ------------------------------------------------------------ VNNTSVSPDG-------------------------------------------------- VNNTSVSPDGKLLAVLGDSTECLIADANTGKITGSLKGHLDYSFSSAWHPDGQILATGNQ VNNTSVSPDGKLLAVLGDSTECLIADANTGKITGSLKGHLDYSFSSAWHPDGQILATGNQ ------------------------------------------------------------ ------------------------------------------------------------ VNNTSVSPDGKLLAVLGDSTECLIADANTGKITGSLKGHLDYSFSSAWHPDGQILATGNQ ------------------------------------------------------------ VNHTSVSPDKKLLAVVGDDRDALLVDSRNGKVTSTLVGHLDYSFASAWHLDGVTFATGNQ VNNTSVSPDGKLLAVLGDSSDCLIADSQSGKEMARLKGHLDYSFSSAWHPDGRVVATGNQ xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx ------------------------------------------------------------ ------------------------------------------------------------ ------------------------------------------------------------ ------------------------------------------------------------ DKTCRLWDIRNLSQSMAVLKGRMGAIRALRFTSDGRFLAMAEPADFVHIFDSHSGYEQGQ DKTCRLWDIRNLSQSMAVLKGRMGAIRALRFTSDGRFLAMAEPADFVHIFDSHSGYEQGQ ------------------------------------------------------------ ------------------------------------------------------------ DKTCRLWDIRNLSQSMAVLKGRMGAIRALRFTSDGRFLAMAEPADFVHIFDSHSGYEQGQ ------------------------------------------------------------ DKTCRVWDIRNPSTSLAVLRGNIGAIRCIRYSSDGRFLLFSEPADFVHVYSTAECYRKRQ DRTCRVWDVRNMSRSVAVLEGRIGAVRGLRYSPDGRFLAASEPADFVHVYDAAAGYADAQ xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx ------------------------------------------------------* ------------------------------------------------------* ------------------------------------------------------* ------------------------------------------------------* EIDLFGEIAGISFSPDTEALFVGIADRTYGSLLEFNRKRHYNYLDSF-------* EIDLFGEIAGISFSPDTEALFVGIADRTYGSLLEFNRKRHYNYLDSF-------* ------------------------------------------------------* ------------------------------------------------------* EIDLFGEIAGISFSPDTEALFVGIADRTYGSLLEFNRKRHYNYLDSF-------* ------------------------------------------------------* EIDFFGEISGISLSPDD------ESLFVGVCDRVYASLLNYRLVHANGYLDSYM* EIDLFGEIAGVAFSPAGNNGGGGEALFVSIADRTYGSLLEFHRRRRHGYLDCYV* xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx-------*

The consensus amino acid sequence can be used to identify DNA corresponding to the full scope of this invention that is useful in providing transgenic plants, e.g., corn, soybean and cotton plants with transgenic cells expressing protein encoding DNA that impart an enhanced agronomic traits. For example, enhanced nitrogen use efficiency, enhanced yield, enhanced water use efficiency, enhanced growth under cold stress and/or improved seed compositions are imparted by the expression in the plants of DNA encoding a protein with amino acid sequence identical to the consensus amino acid sequence.

Example 8 Identification of Target Genes of Transcription Factors ABF3 and CBF3 Chemical Kinetics Models to Identify Regulator-Target Relationships.

It has been shown both in mRNA blotting and microarray experiments that activation of regulators under stress conditions usually occurs earlier than that of its targets (Haake, 2002, Seki, 2002a). In eukaryotic cells, the effect of a regulator is usually achieved in multiple steps, including transcription of the regulator genes, transportation of the regulator mRNA(s) out of the nucleus, translation of the transcript(s), transportation of the regulator protein back to the nucleus, and the binding of the regulator protein to the promoter regions of its target genes to achieve transcriptional regulation. Noticeable timing difference exists among changes in concentrations of the regulator mRNA, the regulator protein, and the mRNAs of its targets. A chemical kinetics model naturally fits this context by taking into account of the time lags among these events.

Because the active level of the regulator protein is not measured directly in microarray experiments, the regulator protein concentration is treated as a hidden variable in our model to serve as the link between the measurable mRNA concentrations of a regulator and its target(s). More specifically, the regulator protein concentration can be modeled by the following chemical kinetic equation without considering post-translational regulation:

$\begin{matrix} {\frac{R_{p}}{t} = {K_{tran}R_{m}K_{p}R_{p}}} & {{equation}\mspace{14mu} (1)} \end{matrix}$

where R_(p) is the regulator protein concentration; R_(m) is the regulator mRNA concentration; K_(tran) is the apparent rate of mRNA translation, and K_(p) is the turnover rate of the regulator protein. Accordingly, the time course of the target mRNA concentration can be modeled with the following equation

$\begin{matrix} {\frac{T_{m}}{t} = {B_{t} + {f\left( R_{p} \right)} - {K_{t}T_{m}}}} & {{equation}\mspace{14mu} (2)} \end{matrix}$

where T_(m) is the concentration of the target mRNA; B_(t) is the basal transcription rate of the target gene; and K_(t) is the turnover rate of the target mRNA; f(R_(p)) measures the regulated transcription rate, which is different for activators and repressors. For activators, it has the following Taylor first order approximation when R_(p) is small (Chen et al., 1999).

$\begin{matrix} {{f\left( R_{p} \right)} = \left. {{f\left( {R_{p} = 0} \right)} + \frac{\left( {f\left( R_{p} \right)} \right.}{R_{p}}} \middle| {}_{{Rp} = 0}R_{p} \right.} & {{equation}\mspace{14mu} (3)} \end{matrix}$

f(R_(p)=0) is equal to zero, assuming target gene transcription should not be activated when there is no regulator protein.

$\left. \frac{\left( {f\left( R_{p} \right)} \right.}{R_{p}} \right|_{{Rp} = 0}$

is the activation rate of regulator protein on the target gene. If it is replaced by parameter K_(act) for simplicity, f(R_(p)) takes the following form:

f(R _(p))=K _(act) R _(p)  equation (4)

The basal level target transcription rate should satisfy the following condition:

B _(t) +f(R _(pbasal))−K _(t) T _(mbasal)=0  equation (5)

Where R_(pbasal) and T_(mbasal) are the basal concentrations of the regulator protein and target mRNA, respectively.

Usually, what is reported in transcription profiling experiment is not the absolute concentration of mRNA, but rather a fold change compared to basal transcription level of that gene. Thus, we define relative changes of R_(m) and T_(m) as R_(m)′ and T_(m)′

R _(m) ′=R _(m) /R _(basal)−1  equation (6)

T _(m) ′=T _(m) /T _(mbasal)−1  equation (7)

Combining equation (1), (2), (4), (5), (6) and (7), and considering the fact that K_(tran)R_(mbasel)−K_(p)R_(pbaseal)=0, leads to the following second order ordinary differential equation:

$\begin{matrix} {{\frac{^{2}\left( T_{m}^{\prime} \right)}{t^{2}} + {\left( {K_{t} + K_{p}} \right)\frac{\left( T_{m}^{\prime} \right)}{t}} + {K_{t}K_{p}T_{m}^{\prime}}} = {\gamma \; R_{m}^{\prime}}} & {{equation}\mspace{14mu} (8)} \end{matrix}$

Where γ=K_(act)K_(tran)R_(mbasal)/T_(mbasel)

Given all the model parameters, the relationship between the relative mRNA levels of regulator and its target, R_(m)′ and T_(m)′, is defined by Equation (8). In other words, for the target gene of a regulator, its relative mRNA level T_(m)′ has to satisfy equation (8), given the model parameters and the relative regulator mRNA level R_(m)′. It is interesting to note that the regulator protein concentration, a key variable in the original model equations, is not involved explicitly in the final equation relating the relative mRNA levels of regulator and target. To predict the target of a specific regulator, we can solve equation (8) to obtain the theoretical target behavior curve, and then find the genes with mRNA levels similar to the theoretical curve, which will be identified as the potential targets of that regulator.

In the case of transcript expression profiling experiments under stress conditions, the initial conditions should be the following:

$\begin{matrix} {\left. T_{m}^{\prime} \right|_{t = 0} = 0} & {{equation}\mspace{14mu} (9)} \\ {\left. \frac{\left( T_{m}^{\prime} \right)}{t} \right|_{t = 0} = 0} & {{equation}\mspace{14mu} (10)} \end{matrix}$

Because the target gene mRNA and the regulator protein should be at their basal levels at the onset of stress condition (t=0). It is apparent from equations (2) and (5) that initial condition (10) should be true.

To approximate R_(m), a stepwise linear model can be fit as follows:

R _(m)′_(i)(t)=α_(i)+β_(i) t t _(i) ≦t≦t _(i+1) i=0, . . . , n−1  equation (11)

Where t_(i) is i^(th) time point; and α_(i) and β_(i) are the parameters of stepwise linear function in each time interval, which are determined by the measured regulator mRNA levels at the two adjacent time points. Equation (8) has analytic solution:

Tm _(i)(t)′=A _(i) e ^(−K) ^(t) ^(t) +B _(i) e ^(−K) ^(p) ^(t) +C _(i) +D _(i) t t _(i) ≦t≦t _(i+1) , i=0, . . . , n−1  equation (12)

Where D_(i)=β_(i)γ/K_(p)K_(t) and C_(i)=[α_(i)γ−(K_(p)+K_(t))D_(i)]/K_(p)K_(t) The contiguous restrictions on T_(m)′ are stated in the following equations:

$\begin{matrix} {{{{Tm}_{i}^{\prime}(t)} = {{Tm}_{i + 1}^{\prime}(t)}},{{{when}\mspace{14mu} t} = {{t_{i}\mspace{20mu} i} = 1}},\ldots \mspace{14mu},{n - 1.}} & {{equation}\mspace{14mu} (13)} \\ {{\frac{\left( {{Tm}_{i}^{\prime}(t)} \right)}{t} = \frac{\left( {{Tm}_{i + 1}^{\prime}(t)} \right)}{t}},{{{when}\mspace{14mu} t} = {{t_{i}\mspace{20mu} i} = 1}},\ldots \mspace{14mu},{n - 1.}} & {{equation}\mspace{14mu} (14)} \end{matrix}$

After substituting equation (12) into equations (9), (10), (13) and (14), A_(i) and B_(i) can be obtained by solving sets of linear algebra equations, and are functions of α_(i), β_(i), γ, K_(t) and K_(p).

Learning model parameters. For each regulator and target pair, there are three parameters involved in equation (8), the target mRNA turnover rate K₁, the active regulator turnover rate K_(p), and γ, which is equal to K_(act)K_(tran)R_(basal)/T_(basel). K_(act) represents the strength of regulator protein effect on the target gene; K_(tran) is the translation rate of regulator mRNA. They lump together with the ratio of basal mRNA concentrations of regulator and target to form parameter γ, which determines the magnitude of the relative target mRNA level but not its shape. It is the parameters K_(t) and K_(p) that determine the shape of the relative target mRNA level, such as how fast the target gene responds to the regulator.

For gene expression experiments under stress conditions in plants, the kinetics model can be trained with known regulator-target pair reported in the literature (e.g., CBF and RD17 in Arabidopsis under cold stress) with a non-linear regression model. When the normalized expression profile of a target gene with its maximal response is considered, there is no need to keep γ as a free model parameter (γ1=nγ2 leads to T_(m1)′=nT_(m2)′ when other parameters are kept the same in equations (8), (9) and (10)). Therefore, only two parameters K_(t) and K_(p) are estimated from the non-linear regression model, and are used to predict other regulators and their targets in plant stress response. The theoretical target mRNA expression profiles are calculated for all the genes annotated as transcription factors, and Pearson correlation coefficient is computed for each theoretical target profile and each observed expression profile in each stress condition. When high correlation in one or several stress conditions is found, the transcription factor could be one of the putative regulators of the corresponding gene.

Target gene prediction using promoter motif analysis. As an additional line of evidence for regulator-target pair prediction, we used promoter motif analysis to correlate regulators and their potential targets. Differentially expressed genes under stress conditions measured in microarray experiments can be partitioned into certain number of clusters based on the similarity in their expression profiles. All known promoter motifs within 1500 base-pairs distance to the starting codon were extracted from AGRIS database (Davuluri, 2003) for each gene. The frequency of each promoter motif in each cluster is computed, and Fisher's Exact Test is conducted to test the over-representation of certain promoter motifs. Enriched promoter motifs for a given cluster are selected as putative regulator motifs when statistical significance meets certain cutoff value (e.g., p-value 0.05). When a transcription factor (or a family of transcription factors) is known to bind to the putative regulator motif, the transcription factor(s) should be the putative regulators of target genes with the regulator motif in that cluster.

Combining evidences from kinetics models and promoter analysis. Kinetics models and promoter analysis independently predict putative regulator-target pairs, we attempted to combine their results to enhance our ability to detect true regulator-target pairs. In our kinetics models, for each target gene only the transcription factors with a Pearson correlation coefficient higher than certain cutoff in at least one stress condition are considered as its potential regulators. It is possible that the same regulator regulates its target genes in different stress conditions. Therefore, it is reasonable to give a higher ranking for a regulator if its theoretical target profiles are correlated to those of certain gene in multiple conditions. Based on these ideas, a ranking score for each possible regulator-target pair is derived as follows:

$\begin{matrix} {{{score}\left( {r_{i},t_{j}} \right)} = {\prod\limits_{k}{{R_{k}\left( {r_{i},t_{j}} \right)}/N}}} & {{equation}\mspace{14mu} (15)} \end{matrix}$

Where R_(k)(r_(i),t_(j)) is the rank of Pearson correlation coefficient of the theoretical target profile of transcription factor r_(i) to that of gene t_(j) in stress condition k; N is the total number of transcription factors on DNA chip.

The rank of the scores for putative transcription factors should represent the likelihood of them being the true regulator for a specific gene. Similarly, the rank of p-value of motif enrichment is the indicator of the likelihood of a transcription factor(s) being the true regulator for a specific target. Lastly, we combine both rankings from kinetics model prediction and promoter analysis by defining a score for a given regulator-target pair as following:

$\begin{matrix} {{{L\left( {r_{i},t_{j}} \right)} = {\prod\limits_{m}{{{rank}_{m}\left( {r_{i},t_{j}} \right)}/N}}}\mspace{14mu} {{m = 1},2}} & {{equation}\mspace{14mu} (17)} \end{matrix}$

Where L(r_(i),t_(j)) can be viewed as the strength of transcription factor r_(i) to be the regulator of gene t_(j); rank₁(r_(i),t_(j)) and rank₂(r_(i),t_(j)) are the rank of score(r_(i),t_(j)) from kinetics model prediction, and the rank of p-value of regulator r_(i) binding motifs enrichment for the cluster with gene t_(i), respectively.

This method was applied to an Arabidopsis gene expression dataset measuring responses to various stress conditions (Seki et al., 2002a; Seki, et al., 2002b). In this experiment, wild-type Arabidopsis plants were subject to stress treatments for various periods (1, 2, 5, 10 and 24 hours), and extracted mRNA samples were hybridized to a cDNA microarray with ˜7000 full-length cDNAs. 493 genes were chosen for the analysis, as each of these genes was differentially regulated in at least one of the stress conditions.

TABLE 8 The evidence of the predicted target genes of CBF3. This table shows the evidence strength, whether evidence from kinetics model or enriched promoter analysis exists for each predicted target. Evidence Kinetics Promoter SEQ ID NO Target strength (10⁻⁵) model Analysis / At1g01470 2.13333 yes yes / AtGolS3 2.13333 yes yes / RD17 2.13333 yes yes / ERD10 2.13333 yes yes 175 At1g21790 2.13333 yes yes / ERD7 2.13333 yes yes / cor15A 2.13333 yes yes / FL3-5A3 4.26667 yes yes / kin2 4.26667 yes yes / cor15B 6.4 yes yes 176 ERD4 26.66667 yes no / RD29A 26.66667 yes no / At1g16850 53.33333 yes no 177 and 178 At1g78070 800 no yes / kin1 800 no yes

TABLE 9 The evidence of the predicted target genes of ABF3. This table shows the evidence strength, whether evidence from kinetics model or enriched promoter analysis exists for each predicted target. Evidence Kinetics Promoter SEQ ID NO Target strength (10⁻⁵) model Analysis 179, 180 and 181 At3g47340 1.26222 yes yes 182 At5g13170 1.26222 yes yes 183 At2g19900 1.26222 yes yes 184 and 185 At5g09530 2.52444 yes yes 186 At2g42790 2.52444 yes yes 187 At3g56200 2.52444 yes yes 188 and 189 At5g01520 2.52444 yes yes 190 At5g66780 3.78667 yes yes 191 At5g59320 3.78667 yes yes 192 AtHB7 5.04889 yes yes / RD29B 7.57333 yes yes 193 RD20 7.57333 yes yes

It has been shown that ABF3 and CBF3 confer stress tolerance to transgenic plants. Thus, the target genes of ABF3 and CBF3, identified by this invention, including SEQ ID NO: 368 through SEQ ID NO: 386, and their homologs, are particularly useful for producing transgenic plant cells in crop plants with enhanced stress tolerance.

Example 9 Identification Of Amino Acid Domain by Pfam Analysis

The amino acid sequence of the expressed proteins that were shown to be associated with an enhanced trait were analyzed for Pfam protein family against the current Pfam collection of multiple sequence alignments and hidden Markov models using the HMMER software in the appended computer listing. The Pfam protein families for the proteins of SEQ ID NO: 194 through 386 are shown in Table 10. The Hidden Markov model databases for the identified patent families are also in the appended computer listing allowing identification of other homologous proteins and their cognate encoding DNA to enable the full breadth of the invention for a person of ordinary skill in the art. Certain proteins are identified by a single Pfam domain and others by multiple Pfam domains. For instance, the protein with amino acids of SEQ ID NO: 194 is characterized by three Pfam domains, i.e. PPDK_N, PEP-utilizer and PEP-utilizer_C.

TABLE 10 PEP SEQ ID Pfam domain NO GENE ID name begin stop score E-value 194 PHE0003351_PMON81242.pep PPDK_N 99 464 710.9 7.90E−211 194 PHE0003351_PMON81242.pep PEP-utilizers 500 601 182.3 1.10E−51 194 PHE0003351_PMON81242.pep PEP-utilizers_C 613 969 723.9 1.00E−214 195 PHE0003351_PMON83625.pep PPDK_N 99 464 710.9 7.90E−211 195 PHE0003351_PMON83625.pep PEP-utilizers 500 601 182.3 1.10E−51 195 PHE0003351_PMON83625.pep PEP-utilizers_C 613 969 723.9 1.00E−214 196 PHE0000207_PMON77878.pep Pkinase 1 259 343.1 4.40E−100 197 PHE0000208_PMON77879.pep Pkinase 1 259 353.4 3.30E−103 198 PHE0000209_PMON77891.pep Pkinase 1 259 354.9 1.20E−103 199 PHE0000210_PMON77880.pep Pkinase 1 259 359.4 5.40E−105 200 PHE0001329_PMON92878.pep Pkinase 12 266 354.3 1.80E−103 200 PHE0001329_PMON92878.pep NAF 311 371 123.6 5.10E−34 201 PHE0001425_PMON79162.pep CAF1 19 252 368.1 1.30E−107 202 PHE0001573_PMON92870.pep GATase_2 2 162 55.5 3.10E−15 202 PHE0001573_PMON92870.pep Asn_synthase 210 451 329.6 5.00E−96 203 PHE0001664_PMON99280.pep FAD_binding_4 69 213 83.4 6.30E−22 204 PHE0001674_PMON79194.pep Myb_DNA-binding 25 70 36.3 9.90E−08 205 PHE0002026_PMON96489.pep Ammonium_transp 36 459 628.5 5.10E−186 206 PHE0002108_PMON92821.pep CSD 1 65 155.1 1.60E−43 207 PHE0002109_PMON93856.pep CSD 1 67 144.8 2.10E−40 208 PHE0002508_PMON92607.pep CBFD_NFYB_HMF 24 89 130.9 3.20E−36 209 PHE0002650_PMON81832.pep SRF-TF 9 59 106.9 5.50E−29 209 PHE0002650_PMON81832.pep K-box 73 172 118.4 1.90E−32 210 PHE0002989_PMON95630.pep Miro 10 126 74.3 3.40E−19 210 PHE0002989_PMON95630.pep Ras 11 173 288.8 9.30E−84 212 PHE0003300_PMON95106.pep MtN3_slv 12 99 131.1 2.80E−36 212 PHE0003300_PMON95106.pep MtN3_slv 133 219 134.9 2.00E−37 214 PHE0003389_PMON94682.pep p450 48 527 286.5 4.60E−83 215 PHE0003614_PMON95111.pep Pyridoxal_deC 33 381 531.8 6.80E−157 216 PHE0003684_PMON92807.pep Myb_DNA-binding 118 168 47.9 3.00E−11 217 PHE0003684_PMON93378.pep Myb_DNA-binding 118 168 47.9 3.00E−11 218 PHE0003853_PMON92602.pep Cyclin_N 46 171 72.6 1.10E−18 219 PHE0003903_PMON98271.pep TPP_enzyme_N 44 220 302.9 5.50E−88 219 PHE0003903_PMON98271.pep TPP_enzyme_M 241 390 157.3 3.70E−44 220 PHE0003905_PMON99283.pep Aldedh 30 492 514.4 1.10E−151 221 PHE0003907_PMON98066.pep Ribosomal_L12 124 191 62.6 1.20E−15 222 PHE0003908_PMON98064.pep DnaJ 31 93 128.9 1.30E−35 223 PHE0003960_PMON95079.pep CTP_transf_2 56 186 142.9 7.70E−40 224 PHE0003967_PMON95088.pep GST_N 11 84 43.3 7.40E−10 228 PHE0004023_PMON92446.pep PHD 198 248 54.9 2.40E−13 229 PHE0004026_PMON93885.pep Aa_trans 44 438 409.4 4.80E−120 230 PHE0004027_PMON93860.pep FAD_binding_4 64 218 83.9 4.60E−22 231 PHE0004028_PMON94697.pep Alpha-amylase 10 426 −62.1 4.30E−06 232 PHE0004034_PMON92631.pep DUF1336 236 478 491.8 7.30E−145 234 PHE0004047_PMON92619.pep LIM 11 68 53.4 7.00E−13 234 PHE0004047_PMON92619.pep LIM 110 167 63.9 4.70E−16 235 PHE0004047_PMON93388.pep LIM 11 68 53.4 7.00E−13 235 PHE0004047_PMON93388.pep LIM 110 167 63.9 4.70E−16 236 PHE0004068_PMON93663.pep AWPM-19 1 125 287.6 2.20E−83 237 PHE0004071_PMON93311.pep RRM_1 105 174 77.3 4.40E−20 238 PHE0004072_PMON93654.pep MMR_HSR1 214 324 65.7 1.40E−16 239 PHE0004072_PMON93669.pep MMR_HSR1 214 324 65.7 1.40E−16 241 PHE0004075_PMON92851.pep MtN3_slv 15 104 75.5 1.50E−19 241 PHE0004075_PMON92851.pep MtN3_slv 137 223 97.2 4.50E−26 242 PHE0004080_PMON93321.pep peroxidase 19 227 241 2.40E−69 243 PHE0004084_PMON95141.pep Phi_1 35 314 691.3 6.40E−205 244 PHE0004093_PMON93332.pep Dimerisation 40 100 105.7 1.20E−28 244 PHE0004093_PMON93332.pep Methyltransf_2 104 350 317.5 2.20E−92 245 PHE0004093_PMON94155.pep Dimerisation 40 100 105.7 1.20E−28 245 PHE0004093_PMON94155.pep Methyltransf_2 104 350 317.5 2.20E−92 247 PHE0004144_PMON93842.pep Cofilin_ADF 16 143 152.4 1.10E−42 248 PHE0004148_PMON92574.pep Iso_dh 28 412 521.5 8.40E−154 249 PHE0004149_PMON92471.pep HEAT_PBS 115 143 23.8 0.00057 249 PHE0004149_PMON92471.pep HEAT_PBS 155 181 37.8 3.50E−08 249 PHE0004149_PMON92471.pep HEAT_PBS 186 212 22.4 0.0015 249 PHE0004149_PMON92471.pep HEAT_PBS 260 287 16.8 0.074 250 PHE0004149_PMON93899.pep HEAT_PBS 115 143 23.8 0.00057 250 PHE0004149_PMON93899.pep HEAT_PBS 155 181 37.8 3.50E−08 250 PHE0004149_PMON93899.pep HEAT_PBS 186 212 22.4 0.0015 250 PHE0004149_PMON93899.pep HEAT_PBS 260 287 16.8 0.074 251 PHE0004152_PMON93672.pep AT_hook 69 81 7.4 1.1 251 PHE0004152_PMON93672.pep DUF296 96 217 175.1 1.60E−49 252 PHE0004155_PMON92626.pep ADH_N 41 152 176.1 8.20E−50 252 PHE0004155_PMON92626.pep ADH_zinc_N 181 324 127.8 2.70E−35 253 PHE0004156_PMON92623.pep NPH3 135 364 219.3 7.80E−63 254 PHE0004162_PMON92481.pep AUX_IAA 22 279 395.9 5.30E−116 255 PHE0004164_PMON92465.pep X8 29 115 168.4 1.70E−47 257 PHE0004167_PMON93333.pep APS_kinase 108 264 363.4 3.20E−106 258 PHE0004168_PMON93855.pep FAD_binding_4 84 225 93 8.10E−25 258 PHE0004168_PMON93855.pep BBE 476 534 120.1 5.70E−33 259 PHE0004169_PMON92568.pep Aldo_ket_red 14 298 389.4 4.80E−114 260 PHE0004184_PMON92565.pep UIM 214 231 14.4 0.29 260 PHE0004184_PMON92565.pep UIM 298 315 24 0.00049 261 PHE0004185_PMON92802.pep p450 39 504 157.8 2.70E−44 262 PHE0004188_PMON92803.pep HSF_DNA-bind 70 233 177.8 2.50E−50 263 PHE0004190_PMON92801.pep HLH 175 213 9.2 0.014 264 PHE0004208_PMON92834.pep Myb_DNA-binding 5 56 39.1 1.30E−08 264 PHE0004208_PMON92834.pep Myb_DNA-binding 134 181 44.6 3.10E−10 265 PHE0004215_PMON92827.pep PBP 14 170 30 1.50E−07 266 PHE0004223_PMON92840.pep Fasciclin 40 179 11.7 0.00032 266 PHE0004223_PMON92840.pep Fasciclin 217 353 104.9 2.20E−28 267 PHE0004225_PMON94167.pep Aldedh 77 539 880.7 6.30E−262 268 PHE0004226_PMON95114.pep Aldedh 77 539 873 1.30E−259 269 PHE0004227_PMON92605.pep UPF0057 5 55 76.3 8.50E−20 270 PHE0004229_PMON92867.pep UPF0057 4 54 96.4 8.00E−26 271 PHE0004233_PMON92843.pep HSF_DNA-bind 60 236 255.5 1.00E−73 272 PHE0004237_PMON93673.pep HSP20 48 153 184.3 2.80E−52 273 PHE0004243_PMON92621.pep CBFD_NFYB_HMF 22 87 122.5 1.10E−33 274 PHE0004244_PMON92858.pep CBFD_NFYB_HMF 39 104 121 3.00E−33 275 PHE0004245_PMON93813.pep CBFD_NFYB_HMF 25 90 129.2 1.00E−35 276 PHE0004248_PMON94672.pep CBFD_NFYB_HMF 37 102 125.3 1.60E−34 278 PHE0004250_PMON92881.pep CBFD_NFYB_HMF 25 90 119.7 7.80E−33 279 PHE0004252_PMON92606.pep CBFD_NFYB_HMF 14 79 94.1 3.80E−25 280 PHE0004253_PMON92874.pep CBFD_NFYB_HMF 7 71 83.8 5.00E−22 281 PHE0004258_PMON93385.pep Pkinase 5 276 144.7 2.30E−40 282 PHE0004258_PMON93806.pep Pkinase 5 276 144.7 2.30E−40 283 PHE0004259_PMON93384.pep Abhydrolase_3 95 319 302.4 7.70E−88 285 PHE0004261_PMON93389.pep Pkinase 31 282 289.6 5.30E−84 285 PHE0004261_PMON93389.pep Pkinase_Tyr 31 280 69.3 6.10E−20 286 PHE0004261_PMON93655.pep Pkinase 31 282 289.6 5.30E−84 286 PHE0004261_PMON93655.pep Pkinase_Tyr 31 280 69.3 6.10E−20 287 PHE0004262_PMON92862.pep Pkinase 86 366 153.9 3.70E−43 287 PHE0004262_PMON92862.pep Pkinase_Tyr 86 366 132.2 1.30E−36 288 PHE0004262_PMON93360.pep Pkinase 86 366 153.9 3.70E−43 288 PHE0004262_PMON93360.pep Pkinase_Tyr 86 366 132.2 1.30E−36 289 PHE0004264_PMON92845.pep PMEI 25 174 138.8 1.40E−38 290 PHE0004264_PMON93354.pep PMEI 25 174 138.8 1.40E−38 291 PHE0004265_PMON92873.pep Suc_Fer-like 59 308 60.7 4.30E−15 292 PHE0004265_PMON93807.pep Suc_Fer-like 59 308 60.7 4.30E−15 293 PHE0004266_PMON92877.pep Myb_DNA-binding 298 348 46.6 7.80E−11 294 PHE0004284_PMON93857.pep U-box 23 97 98.2 2.30E−26 295 PHE0004285_PMON95136.pep CBFD_NFYB_HMF 61 126 123 7.70E−34 296 PHE0004286_PMON93666.pep ICL 21 551 1239.3 0 297 PHE0004287_PMON93344.pep ICL 21 552 1169.2 0 298 PHE0004307_PMON94102.pep RWP-RK 196 247 90.7 4.00E−24 299 PHE0004314_PMON93397.pep zf-C3HC4 148 185 34.1 4.50E−07 300 PHE0004321_PMON93811.pep Redoxin 64 228 4.9 0.0016 300 PHE0004321_PMON93811.pep GSHPx 73 181 246.5 5.10E−71 301 PHE0004321_PMON93834.pep Redoxin 64 228 4.9 0.0016 301 PHE0004321_PMON93834.pep GSHPx 73 181 246.5 5.10E−71 302 PHE0004325_PMON93818.pep CcmH 1 139 16.6 6.50E−09 303 PHE0004335_PMON93850.pep DZC 158 193 81.9 1.80E−21 303 PHE0004335_PMON93850.pep DZC 308 343 80.9 3.70E−21 304 PHE0004336_PMON93858.pep DZC 179 214 78.3 2.20E−20 304 PHE0004336_PMON93858.pep DZC 369 404 71.4 2.60E−18 306 PHE0004348_PMON93810.pep CSD 1 65 136.8 5.50E−38 307 PHE0004349_PMON93812.pep CSD 1 65 141.9 1.50E−39 308 PHE0004350_PMON93826.pep CSD 1 66 148.4 1.70E−41 309 PHE0004351_PMON93821.pep CSD 1 66 149.5 8.10E−42 310 PHE0004352_PMON93824.pep CSD 2 68 151.2 2.50E−42 312 PHE0004393_PMON94192.pep efhand 29 57 18 0.031 312 PHE0004393_PMON94192.pep efhand 66 94 25.3 0.0002 312 PHE0004393_PMON94192.pep efhand 110 138 24.2 0.00042 313 PHE0004395_PMON94145.pep C2 16 138 72.1 1.60E−18 313 PHE0004395_PMON94145.pep PLDc 357 392 30.1 7.20E−06 313 PHE0004395_PMON94145.pep PLDc 702 729 37.1 5.70E−08 314 PHE0004396_PMON94137.pep Orn_Arg_deC_N 118 393 282.3 8.80E−82 314 PHE0004396_PMON94137.pep Orn_DAP_Arg_deC 396 596 161.7 1.70E−45 315 PHE0004417_PMON94190.pep Spermine_synth 13 256 516.1 3.40E−152 316 PHE0004418_PMON94368.pep Amino_oxidase 18 504 275.8 7.90E−80 317 PHE0004419_PMON95100.pep Amidohydro_1 95 446 56.2 1.00E−13 317 PHE0004419_PMON95100.pep Amidohydro_3 95 444 −49.9 0.00024 318 PHE0004421_PMON95120.pep AP2 52 118 92.2 1.40E−24 319 PHE0004422_PMON95123.pep AP2 58 123 78.5 1.90E−20 322 PHE0004432_PMON94112.pep Lactamase_B 63 262 81.6 2.20E−21 322 PHE0004432_PMON94112.pep RMMBL 400 440 33.6 6.10E−07 323 PHE0004472_PMON94115.pep Sina 5 205 188 2.00E−53 324 PHE0004472_PMON94126.pep Sina 5 205 188 2.00E−53 325 PHE0004488_PMON95609.pep Anti-silence 1 155 392.9 4.40E−115 327 PHE0004492_PMON95614.pep NPH3 193 435 469.9 2.90E−138 328 PHE0004545_PMON95117.pep Ribosomal_L14 49 196 105.5 1.50E−28 329 PHE0004574_PMON94433.pep Transaldolase 102 405 620.7 1.20E−183 329 PHE0004574_PMON94433.pep efhand 444 472 21.3 0.0031 330 PHE0004606_PMON95627.pep Metallophos 54 249 154 3.60E−43 331 PHE0004620_PMON94189.pep PFK 6 281 515.1 7.30E−152 332 PHE0004620_PMON94442.pep PFK 6 281 515.1 7.30E−152 333 PHE0004622_PMON95621.pep F-box 2 49 43.2 8.10E−10 333 PHE0004622_PMON95621.pep LRR_2 150 175 41.5 2.60E−09 333 PHE0004622_PMON95621.pep FBD 332 382 74.8 2.50E−19 334 PHE0004626_PMON95101.pep Aminotran_3 79 434 323.4 3.50E−94 335 PHE0004630_PMON94367.pep Iso_dh 40 363 326.9 3.20E−95 336 PHE0004634_PMON94385.pep AP2 28 91 114.2 3.40E−31 337 PHE0004640_PMON95066.pep FAE1_CUT1_RppA 75 365 539.9 2.40E−159 337 PHE0004640_PMON95066.pep Chal_sti_synt_C 322 466 8.7 0.0003 337 PHE0004640_PMON95066.pep ACP_syn_III_C 382 464 26.7 2.30E−08 338 PHE0004645_PMON94655.pep 14-3-3 5 241 304.8 1.50E−88 339 PHE0004645_PMON94685.pep 14-3-3 5 241 304.8 1.50E−88 342 PHE0004650_PMON94686.pep Skp1_POZ 4 64 105.3 1.70E−28 342 PHE0004650_PMON94686.pep Skp1 112 190 173 6.90E−49 343 PHE0004652_PMON94657.pep UPF0005 31 247 55.1 2.20E−13 344 PHE0004652_PMON94687.pep UPF0005 31 247 55.1 2.20E−13 346 PHE0004689_PMON95131.pep Pkinase 12 291 357 2.70E−104 347 PHE0004691_PMON95129.pep Spermine_synth 33 278 501.4 9.60E−148 348 PHE0004719_PMON94698.pep zf-C3HC4 203 243 26.6 8.00E−05 349 PHE0004719_PMON95089.pep zf-C3HC4 203 243 26.6 8.00E−05 350 PHE0004734_PMON94667.pep KOW 26 62 30.7 4.80E−06 350 PHE0004734_PMON94667.pep eIF-5a 84 153 125.8 1.10E−34 351 PHE0004735_PMON95116.pep KOW 26 62 32.2 1.70E−06 351 PHE0004735_PMON95116.pep eIF-5a 84 153 120.3 5.10E−33 352 PHE0004739_PMON95110.pep Miro 7 121 68.7 1.70E−17 352 PHE0004739_PMON95110.pep Ras 8 179 270.9 2.30E−78 353 PHE0004753_PMON95105.pep Aldedh 61 520 791.8 3.50E−235 355 PHE0004770_PMON95122.pep DUF1242 2 70 118.4 1.80E−32 357 PHE0004774_PMON95147.pep zf-A20 14 38 33.1 9.10E−07 357 PHE0004774_PMON95147.pep zf-AN1 92 132 68.2 2.50E−17 358 PHE0004777_PMON95118.pep RNA_pol_L 6 83 75.9 1.20E−19 359 PHE0004785_PMON95057.pep Ribosomal_L18p 26 172 251.4 1.70E−72 360 PHE0004786_PMON95604.pep Phi_1 35 314 691.3 6.40E−205 361 PHE0004788_PMON95092.pep DS 53 369 587.4 1.30E−173 362 PHE0004799_PMON95602.pep DAO 34 481 −14.1 7.30E−05 362 PHE0004799_PMON95602.pep Amino_oxidase 42 483 342.7 5.60E−100 363 PHE0004841_PMON95636.pep DNA_photolyase 18 190 254.3 2.30E−73 363 PHE0004841_PMON95636.pep FAD_binding_7 223 501 503 3.20E−148 367 PHE0004888_PMON95603.pep Globin 7 134 73 8.40E−19 367 PHE0004888_PMON95603.pep FAD_binding_6 156 263 29 4.80E−07 367 PHE0004888_PMON95603.pep NAD_binding_1 276 393 13.4 9.80E−05 369 ERD4.pep DUF221 295 710 245.3 1.20E−70 370 At1g78070.2.pep WD40 310 347 34.1 4.40E−07 372 At3g47340.1.pep GATase_2 2 161 99.6 8.60E−27 372 At3g47340.1.pep Asn_synthase 209 450 344.3 1.80E−100 373 At3g47340.3.pep GATase_2 2 161 99.6 8.60E−27 373 At3g47340.3.pep Asn_synthase 209 430 286.1 6.20E−83 374 At3g47340.2.pep GATase_2 2 161 99.6 8.60E−27 374 At3g47340.2.pep Asn_synthase 209 450 344.3 1.80E−100 375 At5g13170.1.pep MtN3_slv 12 99 135.1 1.80E−37 375 At5g13170.1.pep MtN3_slv 134 220 135.4 1.40E−37 376 At2g19900.1.pep malic 107 295 407.1 2.20E−119 376 At2g19900.1.pep Malic_M 297 550 466.9 2.30E−137 379 At2g42790.1.pep Citrate_synt 93 461 506.2 3.40E−149 380 At3g56200.1.pep Aa_trans 21 426 106.3 8.10E−29 381 At5g01520.1.pep zf-C3HC4 146 183 26.1 0.00011 384 At5g59320.1.pep Tryp_alpha_amyl 27 111 114.7 2.30E−31 385 AtHB7.pep Homeobox 30 86 66.6 7.10E−17 385 AtHB7.pep HALZ 87 131 39.2 1.30E−08 386 RD20.pep Caleosin 54 227 469 5.30E−138

TABLE 11 Pfam domain accession gathering name number cutoff domain description 14-3-3 PF00244.9 25 14-3-3 protein ACP_syn_III_C PF08541.1 −24.4 3-Oxoacyl-[acyl-carrier-protein (ACP)] synthase III C terminal ADH_N PF08240.2 −14.5 Alcohol dehydrogenase GroES-like domain ADH_zinc_N PF00107.16 23.8 Zinc-binding dehydrogenase AP2 PF00847.9 0 AP2 domain APS_kinase PF01583.9 25 Adenylylsulphate kinase AT_hook PF02178.8 3.6 AT hook motif AUX_IAA PF02309.6 −83 AUX/IAA family AWPM-19 PF05512.1 25 AWPM-19-like family Aa_trans PF01490.7 −128.4 Transmembrane amino acid transporter protein Abhydrolase_3 PF07859.2 25.8 alpha/beta hydrolase fold Aldedh PF00171.11 −209.3 Aldehyde dehydrogenase family Aldo_ket_red PF00248.10 −97 Aldo/keto reductase family Alpha-amylase PF00128.12 −93 Alpha amylase, catalytic domain Amidohydro_1 PF01979.8 −37.4 Amidohydrolase family Amidohydro_3 PF07969.1 −65.5 Amidohydrolase family Amino_oxidase PF01593.12 −11.4 Flavin containing amine oxidoreductase Aminotran_3 PF00202.10 −207.6 Aminotransferase class-III Ammonium_transp PF00909.10 −144 Ammonium Transporter Family Anti-silence PF04729.4 25 Anti-silencing protein, ASF1-like Asn_synthase PF00733.10 −52.8 Asparagine synthase BBE PF08031.1 25 Berberine and berberine like C2 PF00168.18 3.7 C2 domain CAF1 PF04857.8 −100.5 CAF1 family ribonuclease CBFD_NFYB_HMF PF00808.12 18.4 Histone-like transcription factor (CBF/NF-Y) and archaeal histone CSD PF00313.12 −0.3 ‘Cold-shock’ DNA-binding domain CTP_transf_2 PF01467.16 −11.8 Cytidylyltransferase Caleosin PF05042.3 25 Caleosin related protein CcmH PF03918.4 −30.8 Cytochrome C biogenesis protein Chal_sti_synt_C PF02797.5 −6.1 Chalcone and stilbene synthases, C-terminal domain Citrate_synt PF00285.10 −101.5 Citrate synthase Cofilin_ADF PF00241.10 −4.7 Cofilin/tropomyosin-type actin-binding protein Cyclin_N PF00134.13 −14.7 Cyclin, N-terminal domain DAO PF01266.12 −35.9 FAD dependent oxidoreductase DNA_photolyase PF00875.8 26.1 DNA photolyase DS PF01916.7 −95.2 Deoxyhypusine synthase DUF1242 PF06842.1 25 Protein of unknown function (DUF1242) DUF1336 PF07059.2 −78.2 Protein of unknown function (DUF1336) DUF221 PF02714.5 25 Domain of unknown function DUF221 DUF296 PF03479.4 −11 Domain of unknown function (DUF296) DZC PF08381.1 15.3 Disease resistance/zinc finger/chromosome condensation-like region Dimerisation PF08100.1 18.1 Dimerisation domain DnaJ PF00226.19 −8 DnaJ domain F-box PF00646.21 13.6 F-box domain FAD_binding_4 PF01565.12 −8.1 FAD binding domain FAD_binding_6 PF00970.13 −11.4 Oxidoreductase FAD-binding domain FAD_binding_7 PF03441.4 25 FAD binding domain of DNA photolyase FAE1_CUT1_RppA PF08392.1 −192.7 FAE1/Type III polyketide synthase-like protein FBD PF08387.1 25 FBD Fasciclin PF02469.10 4 Fasciclin domain GATase_2 PF00310.10 −106.2 Glutamine amidotransferases class-II GSHPx PF00255.10 −16 Glutathione peroxidase GST_N PF02798.9 14.6 Glutathione S-transferase, N-terminal domain Globin PF00042.11 −8.8 Globin HALZ PF02183.7 17 Homeobox associated leucine zipper HEAT_PBS PF03130.5 15 PBS lyase HEAT-like repeat HLH PF00010.15 8.2 Helix-loop-helix DNA-binding domain HSF_DNA-bind PF00447.7 −70 HSF-type DNA-binding HSP20 PF00011.10 13 Hsp20/alpha crystallin family Homeobox PF00046.18 −4.1 Homeobox domain ICL PF00463.10 −234 Isocitrate lyase family Iso_dh PF00180.10 −97 Isocitrate/isopropylmalate dehydrogenase K-box PF01486.7 0 K-box region KOW PF00467.18 29.1 KOW motif LIM PF00412.11 0 LIM domain LRR_2 PF07723.2 6 Leucine Rich Repeat Lactamase_B PF00753.16 24.6 Metallo-beta-lactamase superfamily MMR_HSR1 PF01926.11 31.2 GTPase of unknown function Malic_M PF03949.4 −143.9 Malic enzyme, NAD binding domain Metallophos PF00149.17 22 Calcineurin-like phosphoesterase Methyltransf_2 PF00891.7 −103.8 O-methyltransferase Miro PF08477.1 28 Miro-like protein MtN3_slv PF03083.5 −0.8 MtN3/saliva family Myb_DNA-binding PF00249.19 2.8 Myb-like DNA-binding domain NAD_binding_1 PF00175.10 −3.9 Oxidoreductase NAD-binding domain NAF PF03822.4 4.5 NAF domain NPH3 PF03000.4 25 NPH3 family Orn_Arg_deC_N PF02784.7 −76 Pyridoxal-dependent decarboxylase, pyridoxal binding domain Orn_DAP_Arg_deC PF00278.12 6.7 Pyridoxal-dependent decarboxylase, C-terminal sheet domain PBP PF01161.9 −20.6 Phosphatidylethanolamine-binding protein PEP-utilizers PF00391.12 10 PEP-utilising enzyme, mobile domain PEP-utilizers_C PF02896.7 −173 PEP-utilising enzyme, TIM barrel domain PFK PF00365.10 −132 Phosphofructokinase PHD PF00628.17 25.9 PHD-finger PLDc PF00614.11 0 Phospholipase D Active site motif PMEI PF04043.5 25 Plant invertase/pectin methylesterase inhibitor PPDK_N PF01326.8 −87 Pyruvate phosphate dikinase, PEP/pyruvate binding domain Phi_1 PF04674.2 25 Phosphate-induced protein 1 conserved region Pkinase PF00069.14 −70.8 Protein kinase domain Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase Pyridoxal_deC PF00282.9 −158.6 Pyridoxal-dependent decarboxylase conserved domain RMMBL PF07521.1 18.5 RNA-metabolising metallo-beta-lactamase RNA_pol_L PF01193.12 16.9 RNA polymerase Rpb3/Rpb11 dimerisation domain RRM_1 PF00076.11 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) RWP-RK PF02042.5 25 RWP-RK domain Ras PF00071.11 −69.9 Ras family Redoxin PF08534.1 −1 Redoxin Ribosomal_L12 PF00542.8 25 Ribosomal protein L7/L12 C-terminal domain Ribosomal_L14 PF00238.9 −8 Ribosomal protein L14p/L23e Ribosomal_L18p PF00861.12 25 Ribosomal L18p/L5e family SRF-TF PF00319.8 11 SRF-type transcription factor (DNA-binding and dimerisation domain) Sina PF03145.6 −48.4 Seven in absentia protein family Skp1 PF01466.8 −2 Skp1 family, dimerisation domain Skp1_POZ PF03931.5 14.9 Skp1 family, tetramerisation domain Spermine_synth PF01564.6 −93.8 Spermine/spermidine synthase Suc_Fer-like PF06999.2 −42.4 Sucrase/ferredoxin-like TPP_enzyme_M PF00205.11 −23.9 Thiamine pyrophosphate enzyme, central domain TPP_enzyme_N PF02776.7 −70 Thiamine pyrophosphate enzyme, N-terminal TPP binding domain Transaldolase PF00923.9 −49 Transaldolase Tryp_alpha_amyl PF00234.10 −4 Protease inhibitor/seed storage/LTP family U-box PF04564.5 10.5 U-box domain UIM PF02809.10 4.1 Ubiquitin interaction motif UPF0005 PF01027.11 −6.7 Uncharacterised protein family UPF0005 UPF0057 PF01679.7 25 Uncharacterized protein family UPF0057 WD40 PF00400.20 21.5 WD domain, G-beta repeat X8 PF07983.3 −28.8 X8 domain eIF-5a PF01287.9 9.6 Eukaryotic initiation factor 5A hypusine, DNA- binding OB fold efhand PF00036.20 17.5 EF hand malic PF00390.8 25 Malic enzyme, N-terminal domain p450 PF00067.11 −105 Cytochrome P450 peroxidase PF00141.12 −10 Peroxidase zf-A20 PF01754.6 25 A20-like zinc finger zf-AN1 PF01428.6 0 AN1-like Zinc finger zf-C3HC4 PF00097.13 16.9 Zinc finger, C3HC4 type (RING finger)

Example 10 Selection of Transgenic Plants with Enhanced Agronomic Traits

This example illustrates identification of plant cells of the invention by screening transgenic plants and seeds for an enhanced trait. Transgenic seed and plants, e.g., with transgenic corn cells in the plants prepared in Example 2, transgenic soybean cells in the plants prepared in Example 3, transgenic cotton cells in the plants prepared in Example 4, and transgenic cells in the plants prepared in Example 6, are screened for enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil as compared to control plants.

A. Selection for Enhanced Nitrogen Use Efficiency

The physiological efficacy of transgenic corn plants (tested as hybrids) can be tested for nitrogen use efficiency (NUE) traits in a high-throughput nitrogen (N) selection method. The collected data are compared to the measurements from wildtype controls using a statistical model to determine if the changes are due to the transgene. Raw data were analyzed by SAS software. Results shown herein are the comparison of transgenic plants relative to the wildtype controls.

(1) Media Preparation for Planting a NUE Protocol

Planting materials used: Metro Mix 200 (vendor: Hummert) Cat. #10-0325, Scotts Micro Max Nutrients (vendor: Hummert) Cat. #07-6330, OS 4⅓″×3⅞″ pots (vendor: Hummert) Cat. #16-1415, OS trays (vendor: Hummert) Cat. #16-1515, Hoagland's macronutrients solution, Plastic 5″ stakes (vendor: Hummert) yellow Cat. #49-1569, white Cat. #49-1505, Labels with numbers indicating material contained in pots. Fill 500 pots to rim with Metro Mix 200 to a weight of ˜140 g/pot. Pots are filled uniformly by using a balancer. Add 0.4 g of Micro Max nutrients to each pot. Stir ingredients with spatula to a depth of 3 inches while preventing material loss.

(2) Planting a NUE Selection in the Greenhouse

(a) Seed Germination—Each pot is lightly watered twice using reverse osmosis purified water. The first watering is scheduled to occur just before planting; and the second watering, after the seed has been planted in the pot. Ten Seeds of each entry (1 seed per pot) are planted to select eight healthy uniform seedlings. Additional wild type controls are planted for use as border rows. Alternatively, 15 seeds of each entry (1 seed per pot) are planted to select 12 healthy uniform seedlings (this larger number of plantings is used for the second, or confirmation, planting). Place pots on each of the 12 shelves in the Conviron growth chamber for seven days. This is done to allow more uniform germination and early seedling growth. The following growth chamber settings are 25° C./day and 22° C./night, 14 hours light and ten hours dark, humidity ˜80%, and light intensity ˜350 μmol/m²/s (at pot level). Watering is done via capillary matting similar to greenhouse benches with duration of ten minutes three times a day.

(b) Seedling transfer—After seven days, the best eight or 12 seedlings for the first or confirmation pass runs, respectively, are chosen and transferred to greenhouse benches. The pots are spaced eight inches apart (center to center) and are positioned on the benches using the spacing patterns printed on the capillary matting. The Vattex matting creates a 384-position grid, randomizing all range, row combinations. Additional pots of controls are placed along the outside of the experimental block to reduce border effects.

Plants are allowed to grow for 28 days under the low N run or for 23 days under the high N run. The macronutrients are dispensed in the form of a macronutrient solution (see composition below) containing precise amounts of N added (2 mM NH₄NO₃ for limiting N selection and 20 mM NH₄NO₃ for high N selection runs). Each pot is manually dispensed 100 ml of nutrient solution three times a week on alternate days starting at eight and ten days after planting for high N and low N runs, respectively. On the day of nutrient application, two 20 min waterings at 05:00 and 13:00 are skipped. The vattex matting should be changed every third run to avoid N accumulation and buildup of root matter. Table 12 shows the amount of nutrients in the nutrient solution for either the low or high nitrogen selection.

TABLE 12 2 mM NH₄NO₃ 20 mM NH₄NO₃ (high (Low Nitrogen Growth Nitrogen Growth Condition, Low N) Condition, High N) Nutrient Stock mL/L mL/L 1 M NH₄N0₃ 2 20 1 M KH₂PO₄ 0.5 0.5 1 M MgSO₄•7H₂O 2 2 1 M CaCl₂ 2.5 2.5 1 M K₂SO₄ 1 1 Note Adjust pH to 5.6 with HCl or KOH

(c) Harvest Measurements and Data Collection—After 28 days of plant growth for low N runs and 23 days of plant growth for high N runs, the following measurements are taken (phenocodes in parentheses): total shoot fresh mass (g) (SFM) measured by Sartorius electronic balance, V6 leaf chlorophyll measured by Minolta SPAD meter (relative units) (LC), V6 leaf area (cm²) (LA) measured by a Li-Cor leaf area meter, V6 leaf fresh mass (g) (LFM) measured by Sartorius electronic balance, and V6 leaf dry mass (g) (LDM) measured by Sartorius electronic balance. Raw data were analyzed by SAS software. Results shown are the comparison of transgenic plants relative to the wildtype controls.

To take a leaf reading, samples were excised from the V6 leaf. Since chlorophyll meter readings of corn leaves are affected by the part of the leaf and the position of the leaf on the plant that is sampled, SPAD meter readings were done on leaf six of the plants. Three measurements per leaf were taken, of which the first reading was taken from a point one-half the distance between the leaf tip and the collar and halfway from the leaf margin to the midrib while two were taken toward the leaf tip. The measurements were restricted in the area from ½ to ¾ of the total length of the leaf (from the base) with approximately equal spacing between them. The average of the three measurements was taken from the SPAD machine.

Leaf fresh mass is recorded for an excised V6 leaf, the leaf is placed into a paper bag. The paper bags containing the leaves are then placed into a forced air oven at 80° C. for 3 days. After 3 days, the paper bags are removed from the oven and the leaf dry mass measurements are taken.

From the collected data, two derived measurements are made: (1) Leaf chlorophyll area (LCA), which is a product of V6 relative chlorophyll content and its leaf area (relative units). Leaf chlorophyll area=leaf chlorophyll×leaf area. This parameter gives an indication of the spread of chlorophyll over the entire leaf area; (2) specific leaf area (LSA) is calculated as the ratio of V6 leaf area to its dry mass (cm²/g dry mass), a parameter also recognized as a measure of NUE.

TABLE 13 PEP Leaf chlorophyll area Leaf chlorophyll Shoot fresh mass SEQ Percent P- Percent P- Percent P- ID NO Construct ID Event ID run change Delta value change Delta value change Delta value 199 PMON77880 ZM_M61363 1 2.2 140.91 0.5093 1.8 0.55 0.4558 8.9 4.39 0.0396 PMON77880 ZM_M61363 2 −6.1 −311.2 0.038 1.6 0.46 0.3878 −4.5 −2.04 0.1861 PMON77880 ZM_M61882 1 22.3 1423.3 0 15.1 4.68 0 38.5 18.94 0 PMON77880 ZM_M61882 2 5.6 288.92 0.0458 8.4 2.44 0 7.8 3.51 0.0108 204 PMON79194 ZM_M47022 1 11.7 0.09 1.00E−04 11.7 0.09 1.00E−04 −8.1 −16.4 2.00E−04 PMON79194 ZM_M47136 1 −10.4 −0.08 4.00E−04 8.3 0.44 2.00E−04 7.4 0.39 0.0011 PMON79194 ZM_M48721 1 −10.9 −685.75 0.0017 7.1 2.2 0.0016 10.5 4.48 0.0036

Nitrogen Use Field Efficacy Assay

Level I. Transgenic plants provided by the present invention are planted in field without any nitrogen source being applied. Transgenic plants and control plants are grouped by genotype and construct with controls arranged randomly within genotype blocks. Each type of transgenic plants are tested by 3 replications and across 5 locations. Nitrogen levels in the fields are analyzed in early April pre-planting by collecting 30 sample soil cores from 0-24″ and 24 to 48″ soil layer. Soil samples are analyzed for nitrate-nitrogen, phosphorus(P), Potassium(K), organic matter and pH to provide baseline values. P, K and micronutrients are applied based upon soil test recommendations.

Level II. Transgenic plants provided by the present invention are planted in field with three levels of nitrogen (N) fertilizer being applied, i.e. low level (0 N), medium level (80 lb/ac) and high level (180 lb/ac). Liquid 28% or 32% UAN (Urea, Ammonium Nitrogen) are used as the N source and apply by broadcast boom and incorporate with a field cultivator with rear rolling basket in the same direction as intended crop rows. Although there is no N applied to the 0 N treatment the soil should still be disturbed in the same fashion as the treated area. Transgenic plants and control plants are grouped by genotype and construct with controls arranged randomly within genotype blocks. Each type of transgenic plants is tested by 3 replications and across 4 locations. Nitrogen levels in the fields are analyzed in early April pre-planting by collecting 30 sample soil cores from 0-24″ and 24 to 48″ soil layer. Soil samples are analyzed for nitrate-nitrogen, phosphorus(P), Potassium(K), organic matter and pH to provide baseline values. P, K and micronutrients are applied based upon soil test recommendations.

B. Selection for Increased Yield

Many transgenic plants of this invention exhibit improved yield as compared to a control plant. Improved yield can result from enhanced seed sink potential, i.e. the number and size of endosperm cells or kernels and/or enhanced sink strength, i.e. the rate of starch biosynthesis. Sink potential can be established very early during kernel development, as endosperm cell number and size are determined within the first few days after pollination.

Much of the increase in corn yield of the past several decades has resulted from an increase in planting density. During that period, corn yield has been increasing at a rate of 2.1 bushels/acre/year, but the planting density has increased at a rate of 250 plants/acre/year. A characteristic of modern hybrid corn is the ability of these varieties to be planted at high density. Many studies have shown that a higher than current planting density should result in more biomass production, but current germplasm does not perform well at these higher densities. One approach to increasing yield is to increase harvest index (HI), the proportion of biomass that is allocated to the kernel compared to total biomass, in high density plantings.

Effective yield selection of enhanced yielding transgenic corn events uses hybrid progeny of the transgenic event over multiple locations with plants grown under optimal production management practices, and maximum pest control. A useful target for improved yield is a 5% to 10% increase in yield as compared to yield produced by plants grown from seed for a control plant. Selection methods may be applied in multiple and diverse geographic locations, for example up to 16 or more locations, over one or more plating seasons, for example at least two planting seasons to statistically distinguish yield improvement from natural environmental effects. It is to plant multiple transgenic plants, positive and negative control plants, and pollinator plants in standard plots, for example 2 row plots, 20 feet long by 5 feet wide with 30 inches distance between rows and a 3 foot alley between ranges. Transgenic events can be grouped by recombinant DNA constructs with groups randomly placed in the field. A pollinator plot of a high quality corn line is planted for every two plots to allow open pollination when using male sterile transgenic events. A useful planting density is about 30,000 plants/acre. High planting density is greater than 30,000 plants/acre, preferably about 40,000 plants/acre, more preferably about 42,000 plants/acre, most preferably about 45,000 plants/acre. Surrogate indicators for yield improvement include source capacity (biomass), source output (sucrose and photosynthesis), sink components (kernel size, ear size, starch in the seed), development (light response, height, density tolerance), maturity, early flowering trait and physiological responses to high density planting, for example at 45,000 plants per acre, for example as illustrated in Table 14 and 15.

TABLE 14 Timing Evaluation Description comments V2-3 Early stand Can be taken any time after germination and prior to removal of any plants. Pollen shed GDU to 50% shed GDU to 50% plants shedding 50% tassel. Silking GDU to 50% silk GDU to 50% plants showing silks. Maturity Plant height Height from soil surface to 10 plants per plot - Yield flag leaf attachment (inches). team assistance Maturity Ear height Height from soil surface to 10 plants per plot - Yield primary ear attachment node. team assistance Maturity Leaves above ear visual scores: erect, size, rolling Maturity Tassel size Visual scores +/− vs. WT Pre-Harvest Final Stand Final stand count prior to harvest, exclude tillers Pre-Harvest Stalk lodging No. of stalks broken below the primary ear attachment. Exclude leaning tillers Pre-Harvest Root lodging No. of stalks leaning >45° angle from perpendicular. Pre-Harvest Stay green After physiological maturity and when differences among genotypes are evident: Scale 1 (90-100% tissue green)-9 (0-19% tissue green). Harvest Grain Yield Grain yield/plot (Shell weight)

TABLE 15 Timing Evaluation Description V8-V12 Chlorophyll V12-VT Ear leaf area V15-15DAP Chl fluorescence V15-15DAP CER 15-25 DAP Carbohydrates sucrose, starch Pre-Harvest 1st internode diameter Pre-Harvest Base 3 internode diameter Pre-Harvest Ear internode diameter Maturity Ear traits diameter, length, kernel number, kernel weight

Electron transport rates (ETR) and CO2 exchange rates (CER): ETR and CER are measured with Li6400LCF (Licor, Lincoln, Nebr.) around V9-R1 stages. Leaf chlorophyll fluorescence is a quick way to monitor the source activity and is reported to be highly correlated with CO₂ assimilation under varies conditions (Photosyn Research, 37: 89-102). The youngest fully expanded leaf or 2 leaves above the ear leaf is measured with actinic light 1500 (with 10% blue light) micromol m⁻² s⁻¹, 28° C., CO2 levels 450 ppm. Ten plants are measured in each event. There are 2 readings for each plant.

A hand-held chlorophyll meter SPAD-502 (Minolta-Japan) is used to measure the total chlorophyll level on live transgenic plants and the wild type counterparts a. Three trifoliates from each plant are analyzed, and each trifoliate were analyzed three times. Then 9 data points are averaged to obtain the chlorophyll level. The number of analyzed plants of each genotype ranges from 5 to 8.

When selecting for yield improvement a useful statistical measurement approach comprises three components, i.e. modeling spatial autocorrelation of the test field separately for each location, adjusting traits of recombinant DNA events for spatial dependence for each location, and conducting an across location analysis. The first step in modeling spatial autocorrelation is estimating the covariance parameters of the semivariogram. A spherical covariance model is assumed to model the spatial autocorrelation. Because of the size and nature of the trial, it is likely that the spatial autocorrelation may change. Therefore, anisotropy is also assumed along with spherical covariance structure. The following set of equations describes the statistical form of the anisotropic spherical covariance model.

${{C\left( {h;\theta} \right)} = {{{vI}\left( {h = 0} \right)} + {{\sigma^{2}\left( {1 - {\frac{3}{2}h} + {\frac{1}{2}h^{3}}} \right)}{I\left( {h < 1} \right)}}}},$

where I(•) is the indicator function, h=√{square root over ({dot over (x)}²+{dot over (y)}²)} and

{dot over (x)}=[cos(ρπ/180)(x ₁ −x ₂)−sin(ρπ/180)(y ₁ −y ₂)]/ω_(x)

{dot over (y)}=[sin(ρπ/180)(x ₁ −x ₂)+cos(ρπ/180)(y ₁ −y ₂)]/ω_(y)

where s₁=(x₁, y₁) are the spatial coordinates of one location and s₂=(x₂, y₂) are the spatial coordinates of the second location. There are 5 covariance parameters, θ=(ν, σ², ρ, ω_(n), ω_(j)), where ν is the nugget effect, σ² is the partial sill, ρ is a rotation in degrees clockwise from north, Ω_(n) is a scaling parameter for the minor axis and ω_(j) is a scaling parameter for the major axis of an anisotropical ellipse of equal covariance. The five covariance parameters that defines the spatial trend will then be estimated by using data from heavily replicated pollinator plots via restricted maximum likelihood approach. In a multi-location field trial, spatial trend are modeled separately for each location.

After obtaining the variance parameters of the model, a variance-covariance structure is generated for the data set to be analyzed. This variance-covariance structure contains spatial information required to adjust yield data for spatial dependence. In this case, a nested model that best represents the treatment and experimental design of the study is used along with the variance-covariance structure to adjust the yield data. During this process the nursery or the seed batch effects can also be modeled and estimated to adjust the yields for any yield parity caused by seed batch differences. After spatially adjusted data from different locations are generated, all adjusted data is combined and analyzed assuming locations as replications. In this analysis, intra and inter-location variances are combined to estimate the standard error of yield from transgenic plants and control plants. Relative mean comparisons are used to indicate statistically significant yield improvements.

C. Selection for Enhanced Water Use Efficiency (WUE)

Described in this example is a high-throughput method for greenhouse selection of transgenic corn plants to wild type corn plants (tested as inbreds or hybrids) for water use efficiency and method for selection transgenic cotton plants for water use efficiency. This selection process imposes 3 drought/re-water cycles on plants over a total period of 15 days after an initial stress free growth period of 11 days. Each cycle consists of 5 days, with no water being applied for the first four days and a water quenching on the 5th day of the cycle. The primary phenotypes analyzed by the selection method are the changes in plant growth rate as determined by height and biomass during a vegetative drought treatment. The hydration status of the shoot tissues following the drought is also measured. The plant height is measured at three time points. The first is taken just prior to the onset drought when the plant is 11 days old, which is the shoot initial height (SIH). The plant height is also measured halfway throughout the drought/re-water regimen, on day 18 after planting, to give rise to the shoot mid-drought height (SMH). Upon the completion of the final drought cycle on day 26 after planting, the shoot portion of the plant is harvested and measured for a final height, which is the shoot wilt height (SWH) and also measured for shoot wilted biomass (SWM). The shoot is placed in water at 40 degree Celsius in the dark. Three days later, the shoot is weighted to give rise to the shoot turgid weight (STM). After drying in an oven for four days, the shoots are weighted for shoot dry biomass (SDM). The shoot average height (SAH) is the mean plant height across the 3 height measurements. The procedure described above may be adjusted for +/−˜one day for each step given the situation.

To correct for slight differences between plants, a size corrected growth value is derived from SIH and SWH. This is the Relative Growth Rate (RGR). Relative Growth Rate (RGR) is calculated for each shoot using the formula [RGR %=(SWH−SIH)/((SWH+SIH)/2)*100]. Relative water content (RWC) is a measurement of how much (%) of the plant was water at harvest. Water Content (RWC) is calculated for each shoot using the formula [RWC %=(SWM−SDM)/(STM−SDM)*100]. Fully watered corn plants of this age run around 98% RWC.

Progeny transgenic plants are selected from a population of transgenic cotton events under specified growing conditions and are compared with control cotton plants. Control cotton plants are substantially the same cotton genotype but without the recombinant DNA, for example, either a parental cotton plant of the same genotype that was not transformed with the identical recombinant DNA or a negative isoline of the transformed plant. Additionally, a commercial cotton cultivar adapted to the geographical region and cultivation conditions, i.e. cotton variety ST474, cotton variety FM 958, and cotton variety Siokra L-23, are used to compare the relative performance of the transgenic cotton plants containing the recombinant DNA. The specified culture conditions are growing a first set of transgenic and control plants under “wet” conditions, i.e. irrigated in the range of 85 to 100 percent of evapotranspiration to provide leaf water potential of −14 to −18 bars, and growing a second set of transgenic and control plants under “dry” conditions, i.e. irrigated in the range of 40 to 60 percent of evapotranspiration to provide a leaf water potential of −21 to −25 bars. Pest control, such as weed and insect control is applied equally to both wet and dry treatments as needed. Data gathered during the trial includes weather records throughout the growing season including detailed records of rainfall; soil characterization information; any herbicide or insecticide applications; any gross agronomic differences observed such as leaf morphology, branching habit, leaf color, time to flowering, and fruiting pattern; plant height at various points during the trial; stand density; node and fruit number including node above white flower and node above crack boll measurements; and visual wilt scoring. Cotton boll samples are taken and analyzed for lint fraction and fiber quality. The cotton is harvested at the normal harvest timeframe for the trial area. Enhanced water use efficiency is indicated by increased yield, improved relative water content, enhanced leaf water potential, increased biomass, enhanced leaf extension rates, and improved fiber parameters.

D. Selection for Growth Under Cold Stress

(1) Cold germination assay—Three sets of seeds are used for the assay. The first set consists of positive transgenic events (F1 hybrid) where the genes of the present invention are expressed in the seed. The second seed set is nontransgenic, wild-type negative control made from the same genotype as the transgenic events. The third set consisted of two cold tolerant and one cold sensitive commercial check lines of corn. All seeds are treated with a fungicide “Captan” (MAESTRO® 80DF Fungicide, Arvesta Corporation, San Francisco, Calif., USA). 0.43 mL Captan is applied per 45 g of corn seeds by mixing it well and drying the fungicide prior to the experiment.

Corn kernels are placed embryo side down on blotter paper within an individual cell (8.9×8.9 cm) of a germination tray (54×36 cm). Ten seeds from an event are placed into one cell of the germination tray. Each tray can hold 21 transgenic events and 3 replicates of wildtype (LH244SDms+LH59), which is randomized in a complete block design. For every event there are five replications (five trays). The trays are placed at 9.7 C for 24 days (no light) in a Convrion growth chamber (Conviron Model PGV36, Controlled Environments, Winnipeg, Canada). Two hundred and fifty milliliters of deionized water are added to each germination tray. Germination counts are taken 10th, 11th, 12th, 13th, 14th, 17th, 19th, 21st, and 24th day after start date of the experiment. Seeds are considered germinated if the emerged radicle size is 1 cm. From the germination counts germination index is calculated.

The germination index is calculated as per:

Germination index=(Σ([T+1−n _(i) ]*[P _(i) −P _(i−i)]))/T

Where T is the total number of days for which the germination assay is performed. The number of days after planting is defined by n. “i” indicated the number of times the germination had been counted, including the current day. P is the percentage of seeds germinated during any given rating. Statistical differences are calculated between transgenic events and wild type control. After statistical analysis, the events that show a statistical significance at the p level of less than 0.1 relative to wild-type controls will advance to a secondary cold selection. The secondary cold screen is conducted in the same manner of the primary selection only increasing the number of repetitions to ten. Statistical analysis of the data from the secondary selection is conducted to identify the events that show a statistical significance at the p level of less than 0.05 relative to wild-type controls.

TABLE 16 Germination Index 1st Run 2nd Run PEP SEQ % P % P ID CONSTRUCT Event Change value Change value 266 PMON92840 MON810, ZM_M106115 28 0.079 15 0.233 PMON92840 MON810, ZM_M107208 58 0.000 24 0.049 PMON92840 MON810, ZM_M107212 36 0.026 34 0.006 PMON92840 MON810, ZM_M107214 53 0.001 26 0.035 PMON92840 MON810, ZM_M107221 29 0.072 −5 0.663 PMON92840 MON810, ZM_M107224 60 0.000 35 0.004 PMON92840 MON810, ZM_M107228 39 0.017 30 0.016 284 PMON92854 MON810, ZM_M103991 28 0.070 9 0.364 PMON92854 MON810, ZM_M104002 35 0.025 8 0.412 PMON92854 MON810, ZM_M105195 27 0.082 10 0.321 PMON92854 MON810, ZM_M105213 30 0.060 21 0.033 PMON92854 MON810, ZM_M105218 74 0.000 49 0.000 PMON92854 MON810, ZM_M105267 43 0.006 28 0.004 PMON92854 MON810, ZM_M106123 −1 0.965 30 0.002

(2) Cold Shock assay—The experimental set-up for the cold shock assay is the same as described in the above cold germination assay except seeds were grown in potted media for the cold shock assay.

The desired numbers of 2.5″ square plastic pots are placed on flats (n=32, 4×8). Pots were filled with Metro Mix 200 soil-less media containing 19:6:12 fertilizer (6 lbs/cubic yard) (Metro Mix, Pots and Flat are obtained from Hummert International, Earth City, Mo.). After planting seeds, pots are placed in a growth chamber set at 23° C., relative humidity of 65% with 12 hour day and night photoperiod (300 uE/m2-min). Planted seeds are watered for 20 minute every other day by sub-irrigation and flats were rotated every third day in a growth chamber for growing corn seedlings.

On the 10^(th) day after planting the transgenic positive and wild-type negative (WT) plants are positioned in flats in an alternating pattern. Chlorophyll fluorescence of plants is measured on the 10^(th) day during the dark period of growth by using a PAM-2000 portable fluorometer as per the manufacturer's instructions (Walz, Germany). After chlorophyll measurements, leaf samples from each event are collected for confirming the expression of genes of the present invention. For expression analysis six V1 leaf tips from each selection are randomly harvested. The flats are moved to a growth chamber set at 5° C. All other conditions such as humidity, day/night cycle and light intensity are held constant in the growth chamber. The flats are sub-irrigated every day after transfer to the cold temperature. On the 4^(th) day chlorophyll fluorescence is measured. Plants are transferred to normal growth conditions after six days of cold shock treatment and allowed to recover for the next three days. During this recovery period the length of the V3 leaf is measured on the 1^(st) and 3^(rd) days. After two days of recovery V2 leaf damage is determined visually by estimating percent of green V2 leaf.

Statistical differences in V3 leaf growth, V2 leaf necrosis and fluorescence during pre-shock and cold shock can be used for estimation of cold shock damage on corn plants.

(3) Early seedling growth assay—Three sets of seeds are used for the experiment. The first set consists of positive transgenic events (F1 hybrid) where the genes of the present invention are expressed in the seed. The second seed set is nontransgenic, wild-type negative control made from the same genotype as the transgenic events. The third seed set consists of two cold tolerant and two cold sensitive commercial check lines of corn. All seeds are treated with a fungicide “Captan”, (3a,4,7,a-tetrahydro-2-[(trichloromethly)thio]-1H-isoindole-1,3(2H)-dione, Drex Chemical Co. Memphis, Tenn.). Captan (0.43 mL) was applied per 45 g of corn seeds by mixing it well and drying the fungicide prior to the experiment.

Seeds are grown in germination paper for the early seedling growth assay. Three 12″×18″ pieces of germination paper (Anchor Paper #SD7606) are used for each entry in the test (three repetitions per transgenic event). The papers are wetted in a solution of 0.5% KNO₃ and 0.1% Thyram.

For each paper fifteen seeds are placed on the line evenly spaced down the length of the paper. The fifteen seeds are positioned on the paper such that the radical would grow downward, for example longer distance to the paper's edge. The wet paper is rolled up starting from one of the short ends. The paper is rolled evenly and tight enough to hold the seeds in place. The roll is secured into place with two large paper clips, one at the top and one at the bottom. The rolls are incubated in a growth chamber at 23° C. for three days in a randomized complete block design within an appropriate container. The chamber is set for 65% humidity with no light cycle. For the cold stress treatment the rolls are then incubated in a growth chamber at 12° C. for twelve days. The chamber is set for 65% humidity with no light cycle.

After the cold treatment the germination papers are unrolled and the seeds that did not germinate are discarded. The lengths of the radicle and coleoptile for each seed are measured through an automated imaging program that automatically collects and processes the images. The imaging program automatically measures the shoot length, root length, and whole seedling length of every individual seedling and then calculates the average of each roll.

After statistical analysis, the events that show a statistical significance at the p level of less than 0.1 relative to wild-type controls will advance to a secondary cold selection. The secondary cold selection is conducted in the same manner of the primary selection only increasing the number of repetitions to five. Statistical analysis of the data from the secondary selection is conducted to identify the events that show a statistical significance at the p level of less than 0.05 relative to wild-type controls.

TABLE 17 PEP root length shoot length total length SEQ ID Percent P- Percent Percent P- NO construct event run change Delta value change Delta P-value change Delta value 204 PMON79194 ZM_M47022 1 10 1.05 0.0801 5 0.38 0.4359 8 1.43 0.1411 PMON79194 ZM_M47022 2 31 3.31 0 44 3.25 0 36 6.56 0 PMON79194 ZM_M47136 1 9 0.96 0.1078 13 1.08 0.0304 11 2.05 0.0372 PMON79194 ZM_M47136 2 35 3.69 0 36 2.65 1.00E−04 35 6.34 0 PMON79194 ZM_M48721 1 14 1.47 0.0153 10 0.84 0.0908 13 2.31 0.0192 PMON79194 ZM_M48721 2 28 2.99 2.00E−04 33 2.47 3.00E−04 30 5.46 0

(4). Cold Field Efficacy Trial

This example sets forth a cold field efficacy trial to identify gene constructs that confer enhanced cold vigor at germination and early seedling growth under early spring planting field conditions in conventional-till and simulated no-till environments. Seeds are planted into the ground around two weeks before local farmers are beginning to plant corn so that a significant cold stress is exerted onto the crop, named as cold treatment. Seeds also are planted under local optimal planting conditions such that the crop has little or no exposure to cold condition, named as normal treatment. The cold field efficacy trials are carried out in five locations, including Glyndon Minn., Mason Mich., Monmouth Ill., Dayton Iowa, Mystic Conn. At each location, seeds are planted under both cold and normal conditions with 3 repetitions per treatment, 20 kernels per row and single row per plot. Seeds are planted 1.5 to 2 inch deep into soil to avoid muddy conditions. Two temperature monitors are set up at each location to monitor both air and soil temperature daily.

Seed emergence is defined as the point when the growing shoot breaks the soil surface. The number of emerged seedling in each plot is counted everyday from the day the earliest plot begins to emerge until no significant changes in emergence occur. In addition, for each planting date, the latest date when emergence is 0 in all plots is also recorded. Seedling vigor is also rated at V3-V4 stage before the average of corn plant height reaches 10 inches, with 1=excellent early growth, 5=Average growth and 9=poor growth. Days to 50% emergence, maximum percent emergence and seedling vigor are calculated using SAS software for the data within each location or across all locations.

E. Screens for Transgenic Plant Seeds with Increased Protein and/or Oil Levels

This example sets forth a high-throughput selection for identifying plant seeds with improvement in seed composition using the Infratec 1200 series Grain Analyzer, which is a near-infrared transmittance spectrometer used to determine the composition of a bulk seed sample. Near infrared analysis is a non-destructive, high-throughput method that can analyze multiple traits in a single sample scan. An NIR calibration for the analytes of interest is used to predict the values of an unknown sample. The NIR spectrum is obtained for the sample and compared to the calibration using a complex chemometric software package that provides a predicted values as well as information on how well the sample fits in the calibration.

Infratec Model 1221, 1225, or 1227 with transport module by Foss North America is used with cuvette, item #1000-4033, Foss North America or for small samples with small cell cuvette, Foss standard cuvette modified by Leon Girard Co. Corn and soy check samples of varying composition maintained in check cell cuvettes are supplied by Leon Girard Co. NIT collection software is provided by Maximum Consulting Inc. Software. Calculations are performed automatically by the software. Seed samples are received in packets or containers with barcode labels from the customer. The seed is poured into the cuvettes and analyzed as received.

TABLE 18 Typical sample(s): Whole grain corn and soybean seeds Analytical time to run method: Less than 0.75 min per sample Total elapsed time per run: 1.5 minute per sample Typical and minimum sample size: Corn typical: 50 cc; minimum 30 cc Soybean typical: 50 cc; minimum 5 cc Typical analytical range: Determined in part by the specific calibration. Corn - moisture 5-15%, oil 5-20%, protein 5-30%, starch 50-75%, and density 1.0-1.3%. Soybean - moisture 5-15%, oil 15-25%, and protein 35-50%.

TABLE 19 Transgenic corn plants have an increased oil level in seeds PEP 2004 Data 2005 Data SEQ Oil Oil ID NO Event Construct Delta Pvalue delta P value 231 ZM_S90572 PMON17730 0.18 0.22 0.25 0.04 ZM_S90588 PMON17730 N/A N/A 0.10 0.33 ZM_S90610 PMON17730 N/A N/A −0.03 0.78 ZM_S90614 PMON17730 0.26 0.08 0.47 0.00 ZM_S90622 PMON17730 −0.03   0.82 0.31 0.00

TABLE 20 Transgenic corn plants have an increased protein level in seeds PEP SEQ 1st Inbred protein trial 2nd Inbred protein trial ID Mean Mean Mean Mean NO Construct Event transgenic control Delta Pvalue transgenic control Delta Pvalue 208 PMON92607 ZM_M106133 14.06 10.39 3.67 0 11.91 10.06 1.84 0.0023 ZM_M106129 16.46 10.39 6.07 0 16.00 10.06 5.94 0 ZM_M105269 15.80 10.39 5.41 0 14.89 10.06 4.83 0 ZM_M105268 14.07 10.39 3.67 0 12.81 10.06 2.74 0 ZM_M104742 12.57 10.39 2.18 0.0064 12.52 10.06 2.46 0 ZM_M104740 14.45 10.39 4.06 0 12.93 10.06 2.86 0 ZM_M104403 12.90 10.39 2.51 0.0017 12.60 10.06 2.53 0 ZM_M104399 13.21 10.39 2.82 0.0004 14.04 10.06 3.98 0 ZM_M104398 14.91 10.39 4.51 0 12.79 10.06 2.73 0 ZM_M104396 12.13 10.39 1.74 0.0289 12.90 10.06 2.83 0 ZM_M104385 13.12 10.39 2.72 0.0007 12.89 10.06 2.82 0 ZM_M104371 12.23 10.39 1.83 0.0213 12.18 10.06 2.12 0.0004 ZM_M104369 13.41 10.39 3.01 0.0002 11.35 10.06 1.29 0.0309 ZM_M103621 12.26 10.39 1.86 0.0191 10.76 10.06 0.69 0.2425 ZM_M106138 13.74 10.39 3.35 0 13.34 10.06 3.28 0

TABLE 21 Transgenic soybean plants have an increased seed oil level PEP SEQ seed oil content seed protein content ID control transgenic control transgenic NO construct event run mean mean delta mean mean delta 231 PMON94697 construct 1 20.0 19.7 −0.3 42.0 42.9 0.9 analysis 2 19.6 20.2 0.6* 43.3 43.6 0.3 3 19.9 20.6 0.7* 42.5 43.1 0.6 GM_A79833 1 20.2 19.7 −0.3 42.0 42.9 0.9 2 19.6 19.9 0.3 43.3 43.5 0.2 GM_A79838 2 19.6 19.8 0.2 43.3 45.2 1.9* GM_A79839 2 19.6 20.4 0.8* 43.3 43.7 0.4 GM_A79857 2 19.6 19.9 0.3 43.3 43.7 0.4 GM_A79859 2 19.6 20.6 1.0* 43.3 43.7 −0.6 GM_A79894 2 19.6 20.4 0.8* 43.3 43.1 −0.2 GM_A79896 2 19.6 19.8 0.2 43.3 44.4 1.1* GM_A79914 2 19.6 20.9 1.3* 43.3 42.7 −0.6 3 19.9 20.6 0.7 42.5 43.4 0.9 GM_A79934 3 19.9 20.3 0.4* 42.5 43.3 0.8* GM_A79936 3 19.9 21.0 1.1* 42.5 42.5 0.0 Data point with “*” indicate a statistically significant delta (the difference between transgenic and control plants). Seed protein or oil is measured as a percentage of total seed composition. 

1. A plant cell with stably integrated, recombinant DNA comprising a promoter that is functional in plant cells and that is operably linked to DNA from a plant, bacteria or yeast that encodes a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam names consisting of FAD_binding_(—)4, MtN3_slv, Homeobox, FAD_binding_(—)6, RWP-RK, PMEI, FAD_binding_(—)7, RRM_(—)1, Transaldolase, RNA_pol_L, WD40, U-box, Cyclin_N, Skp1, Redoxin, DZC, PBP, TPP_enzyme_M, CBFD_NFYB_HMF, TPP_enzyme_N, PFK, Caleosin, Iso_dh, Ribosomal_L18p, Metallophos, zf-A20, Ras, BBE, NAF, PLDc, DUF1242, Pkinase, C2, p450, Pyridoxal_deC, FBD, UPF0005, HEAT_PBS, GST_N, PEP-utilizers, Alpha-amylase, Amino_oxidase, SRF-TF, Phi_(—)1, Malic_M, Tryp_alpha_amyl, GSHPx, Miro, HSF_DNA-bind, DNA_photolyase, Sina, CTP_transf_(—)2, Abhydrolase_(—)3, Chal_sti_synt_C, ACP_syn_III_C, ADH_zinc_N, CSD, Globin, GATase_(—)2, Amidohydro_(—)1, HLH, HALZ, Amidohydro_(—)3, Lactamase_B, HSP20, DAO, DUF296, AT_hook, AWPM-19, Dimerisation, Suc_Fer-like, Methyltransf_(—)2, Aminotran_(—)3, PHD, MMR_HSR1, Aldo_ket_red, zf-AN1, malic, Fasciclin, UPF0057, DUF221, Pkinase_Tyr, DnaJ, Cofilin_ADF, Orn_Arg_deC_N, Skp1_POZ, Asn_synthase, K-box, LRR_(—)2, Ribosomal_L12, Ammonium_transp, Ribosomal_L14, KOW, DUF1336, DS, Aa_trans, CcmH, peroxidase, eIF-5a, Aldedh, PEP-utilizers_C, ADH_N, UIM, NAD_binding_(—)1, zf-C3HC4, Spermine_synth, AUX_IAA, LIM, Anti-silence, X8, Citrate_synt, 14-3-3, RMMBL, efhand, NPH3, CAF1, ICL, FAE1_CUT1_RppA, Orn_DAP_Arg_deC, PPDK_N, Myb_DNA-binding, AP2, F-box, and APS_kinase wherein the Pfam gathering cuttoff for said protein domain families is stated in Table 11; wherein said plant cell is selected from a population of plant cells with said recombinant DNA by screening plants that are regenerated from plant cells in said population and that express said protein for an enhanced trait as compared to control plants that do not have said recombinant DNA; and wherein said enhanced trait is selected from group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.
 2. A plant cell of claim 1 wherein said protein has an amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group of consensus amino acid sequences consisting of the consensus amino acid sequence constructed for SEQ ID NO: 194 and homologs thereof listed in Table 7 through the consensus amino acid sequence constructed for SEQ ID NO:386 and homologs thereof listed in Table
 7. 3. A plant cell of claim 1 wherein said protein is selected from the group of proteins identified in Table
 1. 4. A plant cell of claim 1 further comprising DNA expressing a protein that provides tolerance from exposure to an herbicide applied at levels that are lethal to a wild type of said plant cell.
 5. A plant cell of claim 4 wherein the agent of said herbicide is a glyphosate, dicamba, or glufosinate compound.
 6. A transgenic plant comprising a plurality of the plant cell of claim 1
 7. A transgenic plant of claim 6 which is homozygous for said recombinant DNA.
 8. A transgenic seed comprising a plurality of the plant cell of claim
 1. 9. A transgenic seed of claim 8 from a corn, soybean, cotton, canola, alfalfa, wheat or rice plant.
 10. Non-natural, transgenic corn seed of claim 9 wherein said seed can produce corn plants that are resistant to disease from the Mal de Rio Cuarto virus or the Puccina sorghi fungus or both.
 11. A transgenic pollen grain comprising a haploid derivative of a plant cell of claim
 1. 12. A method for manufacturing non-natural, transgenic seed that can be used to produce a crop of transgenic plants with an enhanced trait resulting from expression of stably-integrated, recombinant DNA comprising a promoter that is (a) functional in plant cells and (b) is operably linked to DNA from a plant, bacteria or yeast that encodes a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam names consisting of FAD_binding_(—)4, MtN3_slv, Homeobox, FAD_binding_(—)6, RWP-RK, PMEI, FAD_binding_(—)7, RRM_(—)1, Transaldolase, RNA_pol_L, WD40, U-box, Cyclin_N, Skp1, Redoxin, DZC, PBP, TPP_enzyme_M, CBFD_NFYB_HMF, TPP_enzyme_N, PFK, Caleosin, Iso_dh, Ribosomal_L18p, Metallophos, zf-A20, Ras, BBE, NAF, PLDc, DUF1242, Pkinase, C2, p450, Pyridoxal_deC, FBD, UPF0005, HEAT_PBS, GST_N, PEP-utilizers, Alpha-amylase, Amino_oxidase, SRF-TF, Phi_(—)1, Malic_M, Tryp_alpha_amyl, GSHPx, Miro, HSF_DNA-bind, DNA_photolyase, Sina, CTP_transf_(—)2, Abhydrolase_(—)3, Chal_sti_synt_C, ACP_syn_III_C, ADH_zinc_N, CSD, Globin, GATase_(—)2, Amidohydro_(—)1, HLH, HALZ, Amidohydro_(—)3, Lactamase_B, HSP20, DAO, DUF296, AT_hook, AWPM-19, Dimerisation, Suc_Fer-like, Methyltransf_(—)2, Aminotran_(—)3, PHD, MMR_HSR1, Aldo_ket_red, zf-AN1, malic, Fasciclin, UPF0057, DUF221, Pkinase_Tyr, DnaJ, Cofilin_ADF, Orn_Arg_deC_N, Skp1_POZ, Asn_synthase, K-box, LRR_(—)2, Ribosomal_L12, Ammonium_transp, Ribosomal_L14, KOW, DUF1336, DS, Aa_trans, CcmH, peroxidase, eIF-5a, Aldedh, PEP-utilizers_C, ADH_N, UIM, NAD_binding_(—)1, zf-C3HC4, Spermine_synth, AUX_IAA, LIM, Anti-silence, X8, Citrate_synt, 14-3-3, RMMBL, efhand, NPH3, CAF1, ICL, FAE1_CUT1_RppA, Orn_DAP_Arg_deC, PPDK_N, Myb_DNA-binding, AP2, F-box, and APS_kinase; wherein the gathering cutoff for said protein domain families is stated in Table 11; and wherein said enhanced trait is selected from the group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil, said method for manufacturing said seed comprising: (a) screening a population of plants for said enhanced trait and said recombinant DNA, wherein individual plants in said population can exhibit said trait at a level less than, essentially the same as or greater than the level that said trait is exhibited in control plants which do not express the recombinant DNA, (b) selecting from said population one or more plants that exhibit the trait at a level greater than the level that said trait is exhibited in control plants, (c) verifying that said recombinant DNA is stably integrated in said selected plants, (d) analyzing tissue of a selected plant to determine the production of a protein having the function of a protein encoded by nucleotides in a sequence of one of SEQ ID NO:1-193; and (e) collecting seed from a selected plant.
 13. A method of claim 12 wherein plants in said population further comprise DNA expressing a protein that provides tolerance to exposure to an herbicide applied at levels that are lethal to wild type plant cells, and wherein said selecting is effected by treating said population with said herbicide.
 14. A method of claim 13 wherein said herbicide comprises a glyphosate, dicamba or glufosinate compound.
 15. A method of claim 12 wherein said selecting is effected by identifying plants with said enhanced trait.
 16. A method of claim 12 wherein said seed is corn, soybean, cotton, alfalfa, wheat or rice seed.
 17. A method of producing hybrid corn seed comprising: acquiring hybrid corn seed from a herbicide tolerant corn plant which also has stably-integrated, recombinant DNA comprising a promoter that is (a) functional in plant cells and (b) is operably linked to DNA that encodes a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam names consisting of FAD_binding_(—)4, MtN3_slv, Homeobox, FAD_binding_(—)6, RWP-RK, PMEI, FAD_binding_(—)7, RRM_(—)1, Transaldolase, RNA_pol_L, WD40, U-box, Cyclin_N, Skp1, Redoxin, DZC, PBP, TPP_enzyme_M, CBFD_NFYB_HMF, TPP_enzyme_N, PFK, Caleosin, Iso_dh, Ribosomal_L18p, Metallophos, zf-A20, Ras, BBE, NAF, PLDc, DUF1242, Pkinase, C2, p450, Pyridoxal_deC, FBD, UPF0005, HEAT_PBS, GST_N, PEP-utilizers, Alpha-amylase, Amino_oxidase, SRF-TF, Phi_(—)1, Malic_M, Tryp_alpha_amyl, GSHPx, Miro, HSF_DNA-bind, DNA_photolyase, Sina, CTP_transf_(—)2, Abhydrolase_(—)3, Chal_sti_synt_C, ACP_syn_III_C, ADH_zinc_N, CSD, Globin, GATase_(—)2, Amidohydro_(—)1, HLH, HALZ, Amidohydro_(—)3, Lactamase_B, HSP20, DAO, DUF296, AT_hook, AWPM-19, Dimerisation, Suc_Fer-like, Methyltransf_(—)2, Aminotran_(—)3, PHD, MMR—HSR1, Aldo_ket_red, zf-AN1, malic, Fasciclin, UPF0057, DUF221, Pkinase_Tyr, DnaJ, Cofilin_ADF, Orn_Arg_deC_N, Skp1_POZ, Asn_synthase, K-box, LRR_(—)2, Ribosomal_L12, Ammonium_transp, Ribosomal_L14, KOW, DUF1336, DS, Aa_trans, CcmH, peroxidase, eIF-5a, Aldedh, PEP-utilizers_C, ADH_N, UIM, NAD_binding_(—)1, zf-C3HC4, Spermine_synth, AUX_IAA, LIM, Anti-silence, X8, Citrate_synt, 14-3-3, RMMBL, efhand, NPH3, CAF1, ICL, FAE1_CUT1_RppA, Orn_DAP_Arg_deC, PPDK_N, Myb_DNA-binding, AP2, F-box, and APS_kinase; (a) wherein the gathering cuttoff for said protein domain families is stated in Table 11; (b) producing corn plants from said hybrid corn seed, wherein a fraction of the plants produced from said hybrid corn seed is homozygous for said recombinant DNA, a fraction of the plants produced from said hybrid corn seed is hemizygous for said recombinant DNA, and a fraction of the plants produced from said hybrid corn seed has none of said recombinant DNA; (c) selecting corn plants which are homozygous and hemizygous for said recombinant DNA by treating with an herbicide; (d) collecting seed from herbicide-treated-surviving corn plants and planting said seed to produce further progeny corn plants; (e) repeating steps (c) and (d) at least once to produce an inbred corn line; (f) crossing said inbred corn line with a second corn line to produce hybrid seed.
 18. A method of selecting a plant comprising cells of claim 1 wherein an immunoreactive antibody is used to detect the presence of said protein in seed or plant tissue.
 19. Anti-counterfeit milled seed having, as an indication of origin, a plant cell of claim
 1. 20. A method of growing a corn, cotton or soybean crop without irrigation water comprising planting seed having plant cells of claim 1 which are selected for enhanced water use efficiency.
 21. A method of claim 20 comprising providing up to 300 millimeters of ground water during the production of said crop.
 22. A plant cell with stably integrated, recombinant DNA comprising a promoter that is functional in plant cells and that is operably linked to DNA from a plant, bacteria or yeast that encodes a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a MtN3_slv Pfam; wherein the Pfam gathering cuttoff for said protein domain −0.8; wherein said plant cell is selected from a population of plant cells with said recombinant DNA by screening plants that are regenerated from plant cells in said population and that express said protein for an enhanced trait as compared to control plants that do not have said recombinant DNA; and wherein said enhanced trait is enhanced seed oil.
 23. A plant cell of claim 22 wherein said protein has an amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group of consensus amino acid sequences consisting of the consensus amino acid sequence constructed for SEQ ID NO: 212 and homologs thereof listed in Table
 7. 24. A transgenic plant comprising a plurality of the plant cell of claim
 22. 25. The transgenic plant of claim 24 which is homozygous for said recombinant DNA.
 26. A transgenic seed comprising a plurality of the plant cell of claim
 22. 27. The transgenic seed of claim 26 from a corn, soybean, cotton or canola plant.
 28. A transgenic pollen grain comprising a haploid derivative of a plant cell of claim
 22. 29. A method for manufacturing non-natural, transgenic seed that can be used to produce a crop of transgenic plants with an enhanced trait resulting from expression of stably-integrated, recombinant DNA comprising a promoter that is (a) functional in plant cells and (b) is operably linked to DNA from a plant, bacteria or yeast that encodes a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by MtN3_slv Pfam; wherein the gathering cutoff for said protein domain is −0.8; and wherein said enhanced trait is enhanced seed oil, said method for manufacturing said seed comprising: (a) screening a population of plants for said enhanced trait and said recombinant DNA, wherein individual plants in said population can exhibit said trait at a level less than, essentially the same as or greater than the level that said trait is exhibited in control plants which do not express the recombinant DNA, (b) selecting from said population one or more plants that exhibit the trait at a level greater than the level that said trait is exhibited in control plants, (c) verifying that said recombinant DNA is stably integrated in said selected plants, (d) analyzing tissue of a selected plant to determine the production of a protein having the function of a protein encoded by nucleotides in a sequence of one of SEQ ID NO:1; and (e) collecting seed from a selected plant.
 30. The method of claim 29 wherein said selecting is effected by identifying plants with said enhanced trait.
 31. The method of claim 29 wherein said seed is corn, soybean, cotton or canola.
 32. A method of producing hybrid corn seed comprising: acquiring hybrid corn seed from a herbicide tolerant corn plant which also has stably-integrated, recombinant DNA comprising a promoter that is (a) functional in plant cells and (b) is operably linked to DNA that encodes a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by MtN3_slv Pfam; (a) wherein the gathering cuttoff for said protein domain is −0.8; (b) producing corn plants from said hybrid corn seed, wherein a fraction of the plants produced from said hybrid corn seed is homozygous for said recombinant DNA, a fraction of the plants produced from said hybrid corn seed is hemizygous for said recombinant DNA, and a fraction of the plants produced from said hybrid corn seed has none of said recombinant DNA; (c) selecting corn plants which are homozygous and hemizygous for said recombinant DNA by treating with an herbicide; (d) collecting seed from herbicide-treated-surviving corn plants and planting said seed to produce further progeny corn plants; (e) repeating steps (c) and (d) at least once to produce an inbred corn line; (f) crossing said inbred corn line with a second corn line to produce hybrid seed.
 33. A method of selecting a plant comprising cells of claim 22 wherein an immunoreactive antibody is used to detect the presence of said protein in seed or plant tissue.
 34. Anti-counterfeit milled seed having, as an indication of origin, a plant cell of claim
 22. 